Pitino, who earned his 650th career win earlier this season against South Florida, picked up this milestone against Cincinnati counterpart Mick Cronin, a former Cardinals assistant in 2002 and 2003.
The Cardinals (25-5, 13-4 Big East) picked up their sixth straight win and drew within one-half game of first-place Georgetown, which plays at Villanova on Wednesday. Louisville followed up last season's victory over the Bearcats in the conference championship game that sparked a Final Four run.
Louisville's trademark defense ignited this victory, as the Cardinals forced Cincinnati (20-10, 8-9) into a season-high 21 turnovers. Seven came during the first 10 minutes of the second half, including Kevin Ware's steal and dunk for a 53-42 lead, part of a 23-4 run that put the game away.
Peyton Siva and Gorgui Dieng added 11 points each for Louisville, which shot 48 percent (28 of 58). The Cardinals scored 23 points off Cincinnati's mistakes, with several coming during that run after the game was tied at 37.
The surge sealed Pitino's 300th win, an achievement marked by a video tribute and the coach offering a hoarse gratitude to the crowd.
Cashmere Wright scored 15 points and Sean Kilpatrick and JaQuan Parker had 12 each for the Bearcats, who shot 18 of 51 from the field (35 percent) in losing for the sixth time in eight games.
It was a typical Big East game defined by defense.
Despite their recent slump, the Bearcats still rank in the top 30 in field goal defense (18th, 38.5 percent) and scoring (28th, 59.2 per game).
Louisville remained one of the top defensive teams, ranking third in steals per game (10.7) and turnover margin (plus-6). The Cardinals were coming off Saturday's 58-52 win over No. 12 Syracuse, where both teams shot 36 percent; Louisville won it by outscoring Syracuse's bench 22-15, hitting a couple more 3-pointers and taking 12 more free throws.
That victory moved Louisville up two spots in the latest poll, and the Cardinals' objective was using familiar home surroundings to boost their offense.
Their plan worked for the most part in the first half, as the Cardinals shot a respectable 44 percent (15 of 34) and outscored the Bearcats 22-8 in the paint. Louisville moved the ball around well and it helped Siva bounce back from a scoreless game at Syracuse, as he scored 11 points on five field goals including a 3-pointer.
Louisville's defense was key, scoring 12 points off 12 turnovers while holding Cincinnati to 11-of-27 shooting (41 percent). But the Cardinals struggled against the Bearcats' 6-of-14 shooting from beyond the arc, helping Cincinnati stay close and trail just 35-31 at halftime.
A 13-9 run over the final 7:12 of the half provided Louisville's cushion, but that disappeared early in the second half as the Cardinals went nearly 5 minutes without a field goal. Cincinnati rallied to tie the game at 37 by the first media timeout.
Louisville followed with a 9-1 run that eventually became a 60-42 lead.