Many animals, including the fruit fly, are sensitive to small differences in ambient temperature. The ability of Drosophila larvae to choose their ideal temperature (18°C) over other comfortable temperatures (19° to 24°C) depends on a thermosensory signaling pathway that includes a heterotrimeric guanine nucleotide-binding protein (G protein), a phospholipase C, and the transient receptor potential TRPA1 channel. We report that mutation of the gene (ninaE) encoding a classical G protein-coupled receptor (GPCR), Drosophila rhodopsin, eliminates thermotactic discrimination in the comfortable temperature range. This role for rhodopsin in thermotaxis toward 18°C was light-independent. Introduction of mouse melanopsin restored normal thermotactic behavior in ninaE mutant larvae. We propose that rhodopsins represent a class of evolutionarily conserved GPCRs that are required for initiating thermosensory signaling cascades.