The Marlins built a three-run lead early, but the Rockies scored four in the fifth off Zambrano to prevent being swept.
Miami won the first two games, overcoming early deficits of three and four runs.
"This game is crazy," manager Ozzie Guillen said after his team fell to 16-6 in May. "They score four in the first game, and we win. They score three in the second game, and we win. We score three and we lose.
"You have to love baseball. That's why this game is a tough game. We're up by three with Carlos on the mound, I have a little smile on my face. All of a sudden, whoops, Miami Marlins baseball, we're back to one."
Alex White (1-3) had dropped six straight decisions dating back to Sept. 10, 2011, against the Reds. The Rockies had their six-game losing streak snapped, while the Marlins lost for the first time in four games.
Hanley Ramirez had a double and two hits, extending his hitting streak to seven games.
Tulowitzki had a big series, driving in four runs on Wednesday, and he had seven RBIs and two homers in the three games.
"It plays really big to center out there," Tulowitzki said of the Marlins' spacious retractable-roof home. "You have to crush it. I hit a couple balls good to left that went out. If you get it good, it's going to go, but you're not going to get any cheap ones."
Zambrano (2-3) had gone seven straight starts without allowing more than three runs. The right-hander was on the verge of extending that streak before things fell apart in the fifth inning.
With two outs and Ramon Hernandez on first, Zambrano issued a single to Marco Scutaro. Miami led, 4-3, and the right-hander was one out away from qualifying for the win.
Detecting a dip in Zambrano's velocity, Guillen paid a visit to the mound to check on the right-hander. When the mound conference ended, Carlos Gonzalez ripped the first pitch into center for an RBI single. Tulowitzki provided the decisive blow, crushing a three-run homer to left on an 86-mph cutter.
Zambrano insists he is fine, and he chalked up the night to being a poor outing.
"It was one of those outings where you didn't have your best stuff, and you tried to go through it," Zambrano said. "Any time the manager or coach is worried about you, that's very important. Ozzie has been in this game a long time.
"Obviously, he saw the gun and he saw just 86 and he went out there wanting to know what was going on. Nothing happened. I just threw a pitch to Tulowitzki, and I shouldn't have thrown that pitch. I have to go with a different pitch."
The five innings were a season low for Zambrano, and the seven runs raised his ERA from 1.96 to 2.85.
"I hope they trust me about it," Guillen said. "He said he was fine. I expect him to be fine. I don't want to leave a guy in there when he's hurt. One pitch, you can blow everything away. You make one pitch and you can hurt yourself. That's not good. My job is to make sure I'm not taking the risk. That's why I have to believe them. I have to trust them. That's the reason I left him in the game."
Zambrano's velocity was in the 86-89 mph range most of the night. Guillen still was concerned at what he was seeing.
"I was kind of worried because his velocity went down," the manager said. "It seemed he was pushing the ball a little. I was seeing if he was OK, or if he was trying to throw strikes. A lot of guys, when they're not throwing strikes, they have a tendency to [guide] the ball. That wasn't his game. His game was being aggressive. His game is being aggressive with his fastball."
Zambrano's next scheduled start is Monday against the Nationals at home.
"I made too many mistakes today. I don't know if that was because I didn't have my best stuff today," the right-hander said. "But it's over with. I have the Nationals on Monday, and it's should be a good series."
The Marlins used a pair of two-out hits to take a first-inning lead. Ramirez doubled and scored on Greg Dobbs' single to right.
In the second inning, Miami tacked on two more runs. Zambrano had an RBI single and Jose Reyes drove in a run with a single.
The three-run lead didn't hold. In the fourth, the Rockies tied it on Tulowitzki's RBI double and sacrifice flies from Jason Giambi and Michael Cuddyer.
Miami went up 4-3 in the fourth on Reyes' bad-hop infield single, which wasn't handled at first by Giambi. Brett Hayes, who doubled, scored.
"It was one of those weird games," Zambrano said. "I didn't have my best stuff today, and obviously, the Rockies were in a position. They're a good team that can hit. They came with a six-game losing streak. They were hungry today, and I was the bite."