One of the sperm guidance mechanisms in mammals is thermotaxis. An intriguing question is what the identities of the thermosensors are. Here we show that they are opsins belonging to the G-protein coupled receptors superfamily, some of which are known for their function in vision. We demonstrate the presence of all opsins in human sperm and we show that inhibitors of different opsin signaling elements perturb thermotaxis. We also demonstrate that the thermotactic responsiveness of spermatozoa retrieved from rhodopsin-knockout mice is only 30% of the responsiveness of their wild-type counterparts. Taken together, the results suggest that opsins work in mammalian spermatozoa as thermosensors for thermotaxis, and that at least two signaling pathways are involved — phospholipase C signaling for at least one of the opsins (melanopsin), and cyclic nucleotide signaling pathways for other opsins.