The X-linked gene cyclin-dependent kinase-like 5 (CDKL5) is mutated in severe neurodevelopmental disorders, including some forms of atypical Rett syndrome, but the function and regulation of CDKL5 protein in neurons remain to be elucidated. Here, we show that CDKL5 binds to the scaffolding protein postsynaptic density (PSD)-95, and that this binding promotes the targeting of CDKL5 to excitatory synapses. Interestingly, this binding is not constitutive, but governed by palmitate cycling on PSD-95. Furthermore, pathogenic mutations that truncate the C-terminal tail of CDKL5 diminish its binding to PSD-95 and synaptic accumulation. Importantly, down-regulation of CDKL5 by RNA interference (RNAi) or interference with the CDKL5–PSD-95 interaction inhibits dendritic spine formation and growth. These results demonstrate a critical role of the palmitoylation-dependent CDKL5–PSD-95 interaction in localizing CDKL5 to synapses for normal spine development and suggest that disruption of this interaction by pathogenic mutations may be implicated in the pathogenesis of CDKL5-related disorders.