Bryant, who has nearly 1.3 million Twitter followers, reprimanded a fan who tweeted "You're gay" to another fan.
In response to the insult that was posted Sunday on his own Twitter feed, Bryant tweeted: "Just letting you know@PacSmoove @pookeo9 that using "your gay" as a way to put someone down ain't ok! #notcool delete that out ur vocab."
Bryant was fined $100,000 in April for using a gay slur in a nationally televised game, an incident that NBA commissioner David Stern called "offensive and inexcusable."
After receiving a technical foul in a game against the San Antonio Spurs, Bryant yelled at referee Bennie Adams before muttering a gay slur while sitting on the bench. The episode was caught on camera during TNT's national telecast.
Bryant apologized a day later, saying that he did not mean to offend anyone.
Following his tweet Sunday, one of Bryant's followers reminded the All-Star about April's incident.
Bryant tweeted in response: "exactly! That wasn't cool and was ignorant on my part. I own it and learn from it and expect the same from others."
Bryant did not address the Twitter spat after Monday's practice. The Lakers host the Phoenix Suns on Tuesday.
ESPNLosAngeles.com's Ramona Shelburne contributed to this report.