The pupillary light response (PLR) controls pupil’s aperture according to the environmental luminosity, maximizing imaging efficiency while minimizing retinal damage. The PLR is present in most vertebrates, and two mechanisms have been described to achieve it: 1) In almost all vertebrate groups, it’s mediated by the Edinger–Westphal nucleus via the ciliary ganglion. 2) Some vertebrates, such as amphibia, fish, reptile and several mammals additionally show an iris-intrinsic mechanism evoked by the photoreceptor melanopsin, present in the sphincter pupillae muscle, whose activation produces muscle contraction. Besides, in mice it has also been suggested that the retina may directly contribute to PLR. Lampreys, belonging to the oldest group of living vertebrates (branched ⁓560 million years ago), show a well-developed nervous system with key neuronal circuits preserved throughout evolution, including all basic components of the visuo-vestibular control of gaze and all basic types of eye movements. The existence of PLR has recently been reported in lampreys, but its mechanisms are unclear. In the present study, we analyzed the PLR mechanisms in lampreys using video tracking, pharmacology and anatomical techniques. Our results show that the PLR isn’t brain-controlled in lampreys, but it is rather mediated synergistically by the iris-intrinsic mechanism and by direct retinal cholinergic projections to the iris. Although data in other vertebrates are scarce, our results suggest that an iris-intrinsic PLR and one mediated by direct projections from the retina appeared early in vertebrate evolution and were conserved in some vertebrate groups, whereas the brain-mediated PLR evolved later.