The ability of UV radiation to stimulate color change in vertebrates is well known; however, the signaling pathway involved is not fully explained. Since nitric oxide (NO) is among the candidates for this role, in this study the participation of NO signaling in the pigment migration induced by UV radiation in melanophores of the crab Chasmagnathus granulatus was investigated. When the NO donor, SIN-1, was incubated with pieces of epidermis, there was an induction of a dose-dependent pigment dispersion (in vitro assays). When male adults were exposed to different doses of UVA and UVB, N(G)-nitro-l-arginine-methyl-ester, an NO synthase (NOS) blocker produced a decrease of the pigment dispersion induced by UV (in vivo assays). However, in similar assays, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, an NO scavenger, decreased only the pigment dispersion induced by UVA. Interestingly, buthionine sulfoximine did not produce any change in pigment dispersion induced by UVA (in vivo assays) and SIN-1 (in vitro assays). Our results using NADPH-diaphorase histochemistry and immunocytochemistry against nNOS indicated the production of NO by epidermal cells. In conclusion, we suggest that NO is a key molecule for the induction of pigment dispersion in the melanophores of Chasmagnthus granulatus, and also that NOS activation is a fundamental step for this process.