Appearing on stage at a piano, Timberlake sang I Would Die 4 U as a video of Prince also singing the hit was projected onto a huge piece of white material.
Rumours had been circulating ahead of the event that the pop star would cover a Prince song, with some even suggesting his performance would feature a hologram of the late singer.
In the end, his stripped-back tribute marked a reflective moment in his otherwise high-octane performance, as the US Bank Stadium in Minnesota was lit up in purple.
Timberlake had begun his performance with a rendition of Filthy, before singing a medley of some of his greatest hits, including SexyBack, Senorita and Cry Me A River.
His show featured an orchestra, large brass brand and dozens of backing dancers, as well as his new band, the Tennessee Kids.
Fans on social media gave a mixed reaction to the performance, with some describing it as an "amazing" show, while others were not as impressed.
Sean Larkin said: "What a half time show! Justin Timberlake is a one of kind talent. That little piece with Prince was so cool for Minnesota!"
And Rhonda Dunn said: "Amazing, awesome, and beautiful #Prince tribute!"
Timberlake's former band N'Sync also congratulated the singer on Twitter, saying: "Well done @jtimberlake you killed it!"
But @RyleeChillson said the performance was "the concert equivalent of one slice of wonder white bread".
And others complained about not being able to hear the performance properly.
Yashar Ali said: "What's going on with Justin's audio?"
Timberlake's show marked his return to the Super Bowl following a controversial performance with Janet Jackson in 2004.
Speculation had been rife that Jackson might join Timberlake on Sunday, 14 years after their nipplegate moment overshadowed events on the field.
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But she wrote on Twitter on Saturday: "To put to rest any speculation or rumours as to whether I will be performing at the Super Bowl tomorrow: I will not.
"Thank you for your support and I do look forward to seeing you all very soon."
It was the third time Timberlake took to the stage at the sporting event, after first performing in 2001 with his N'Sync bandmates and then three years later with Jackson.
His fifth solo album, Man Of The Woods, was released on February 2.
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