The Los Angeles Lakers star's outburst was caught on camera as he reacted to being judged to have committed a foul.
Bryant is shown apparently mouthing the word "...faggot" after failing to get the referee's attention.
The player has apologised and said his action was out of "frustration in the heat of the game".
"What I said last night should not be taken literally," said Bryant, who also punched his chair and tossed a towel following referee Bennie Adams's call.
NBA commissioner David Stern said in a statement that Bryant's comment was "offensive and inexcusable".
"While I'm fully aware that basketball is an emotional game, such a distasteful term should never be tolerated. Accordingly, I have fined Kobe $100,000."
The Human Rights Campaign, a gay civil rights group, said Bryant's "horribly offensive and distasteful" language "perpetuates a culture of discrimination" and called for a fuller apology.
Bryant is one of the highest paid US sportsmen and one of US basketball's most celebrated stars.
US basketball player Kobe Bryant has been fined $100,000 (£61,000) for using a homophobic slur during an NBA game on Tuesday.