Bats are excellent models for studying the molecular basis of sensory adaptation. In Chiroptera, a sensory trade-off has been proposed between the visual and auditory systems, though the extent of this association has yet to be fully examined. To investigate whether variation in visual performance is associated with echolocation, we experimentally assayed the dim-light visual pigment rhodopsin from bat species with differing echolocation abilities. While spectral tuning properties were similar among bats, we found that the rate of decay of their light-activated state was significantly slower in a nonecholocating bat relative to species that use distinct echolocation strategies, consistent with a sensory trade-off hypothesis. We also found that these rates of decay were remarkably slower compared with those of other mammals, likely indicating an adaptation to dim light. To examine whether functional changes in rhodopsin are associated with shifts in selection intensity upon bat Rh1 sequences, we implemented selection analyses using codon-based likelihood clade models. While no shifts in selection were identified in response to diverse echolocation abilities of bats, we detected a significant increase in the intensity of evolutionary constraint accompanying the diversification of Chiroptera. Taken together, this suggests that substitutions that modulate the stability of the light-activated rhodopsin state were likely maintained through intensified constraint after bats diversified, being finely tuned in response to novel sensory specializations. Our study demonstrates the power of combining experimental and computational approaches for investigating functional mechanisms underlying the evolution of complex sensory adaptations.