Treatment of cultured vertebrate neurons with nitric oxide leads to growth-cone collapse, axon retraction and the reconfiguration of axonal microtubules. We show that the light chain of microtubule-associated protein (MAP) 1B is a substrate for S-nitrosylation in vivo, in cultured cells and in vitro. S-nitrosylation occurs at Cys 2457 in the COOH terminus. Nitrosylation of MAP1B leads to enhanced interaction with microtubules and correlates with the inhibition of neuroblastoma cell differentiation. We further show, in dorsal root ganglion neurons, that MAP1B is necessary for neuronal nitric oxide synthase control of growth-cone size, growth-cone collapse and axon retraction. These results reveal an S-nitrosylation-dependent signal-transduction pathway that is involved in regulation of the axonal cytoskeleton and identify MAP1B as a major component of this pathway. We propose that MAP1B acts by inhibiting a microtubule- and dynein-based mechanism that normally prevents axon retraction.