Hoffman, trying to protect a 3-2 lead, walked Pujols with one out before serving up a 2-1 pitch that Holliday drove over the wall in dead center.
"I've had some good at-bats against him," Holliday said after the Cardinals won for the sixth time in their last seven games at Miller Park, outscoring the Brewers 32-12 over that span. "Anytime against the greatest closer of all time, it's a special home run."
Since the Cardinals acquired Holliday on July 24, they have the majors best record at 31-11.
"He went down there and got it," Brewers manager Ken Macha said. "When he hit it, he kind of did a little jump step. So, when they do that, they know they hit it well."
The victory gave the Cardinals their 12th series win in the last 13. The only hiccup was a two-game split with the New York Mets on Aug. 4-5.
"I think our club just refused to get down or frustrated," Cardinals manager Tony La Russa said. "I would pass out roses to everybody on the roster. It isn't the first time we've done that this season."
St. Louis won for the 14th time in their final at-bat and fourth trailing after eight innings.
It was only the third blown save for Hoffman (1-2) this season, but it spoiled what would have been a remarkable victory for Carlos Villanueva. He was forced to fill in when starter Manny Parra had neck spasms after throwing 21 pitches retiring the Cardinals in the first inning.
The injury put an abrupt end to the rematch between John Smoltz and the Brewers left-hander. Last Thursday, Parra won his 10th game with a 4-3 victory over the Cardinals.
Parra said that he had felt the twinge earlier in the day, but it had calmed down by the time the game started.
"It was the pitch I walked Holliday on," he said, describing the feeling as a shooting pain down his neck and into his upper back. "I came in, and they did some tests. I wasn't going to be able to pitch."
His status for the next start is unknown.
"They didn't tell me anything other than that," Parra said.
Prince Fielder's 38th home run, a two-run shot in the bottom of the first, pushed his major league RBI total to 125 and gave Villanueva a 2-0 lead to start the second.
"They are a good team and, unfortunately, we didn't score enough runs tonight," Fielder said. "They are a good team, and good teams do that every now and then."
The right-hander held the Cardinals scoreless over three innings, retired the side in the fourth on swinging strikeouts and matched his career-high with six overall. He had a big advantage because St. Louis had stacked its lineup with right handers to face Parra.
Pujols hit a solo shot off David Weathers in the seventh for his major-league leading 45th home run and cut the Cardinals' deficit to one. Pujols has scored 12 runs in a span of eight straight games.
The Brewers used eight pitchers which tied the club record for most pitchers used in an nine-inning game.
"Our bullpen pitched their heart and soul out," Macha said.
Brewers RF Corey Hart, out since an emergency appendectomy on Aug. 2, was reinstated from the 15-day disabled list. He pinch hit in the seventh and flied out, but stayed in game in right field. ... DeRosa was back in the starting lineup after missing the last three games with a tight back. ... Milwaukee CF Mike Cameron missed his third consecutive game with a tight hamstring. ... Macha never had a losing record in his eight previous years as a manager, four at Oakland and four in the minors. Milwaukee (66-72) could break that string. ... Fielder is one RBI shy of tying the franchise single-season record set by Cecil Cooper (126) in 1983.