The England captain, who again has been linked with an off-season move away from Tottenham, curled home after an indirect free kick in the eighth minute of Saturday’s lunchtime game.
But Bryan Mbeumo scored twice and set up another goal in the second half for Brentford in their 3-1 win in the Premier League.
Tottenham might now struggle to qualify for any European competition for next season, having come into the game in seventh place — the division’s only qualifying spot for the third-tier Europa Conference League.
Brentford stayed in ninth place but are only one point behind Tottenham ahead of the teams’ final games of the season next weekend.
Kane’s 28th league goal this season was perhaps his best of the campaign as he picked out the top corner with a 25-metre shot after Dejan Kulusevski rolled the ball backward with the sole of his foot at a free kick.
It put Kane eight goals behind the league’s top scorer, Erling Haaland.
Kane will be 30 years old by the time next season starts. With only one year left on his contract, he and Tottenham might decide this summer is the time to part ways to give the club a chance to get some money for their star player and for the England captain to get a better opportunity for trophies.
Mbeumo cut in from the left for both of his goals, in the 50th and 62nd minutes, and then led a counter-attack before setting up Yoane Wissa for a composed finish to seal victory for Brentford in the 88th.
Brentford were without Ivan Toney, who was handed an eight-month suspension on Wednesday for breaching betting regulations.
Tottenham end the season at Leeds next weekend, while Brentford host Manchester City.
