Coordinated intracellular and extracellular signaling is critical to synapse development and functional neural circuit wiring. Here, we report that unesterified docosahexaenoic acid (DHA) regulates functional synapse formation in vivovia retinoid X receptor α (Rxra) signaling. Using Rxraconditional knockout (cKO) mice and virus-mediated transient gene expression, we show that endogenous Rxraplays important roles in regulating spinogenesis and excitatory synaptic transmission in cortical pyramidal neurons. We further show that the effects of RXRA are mediated through its DNA-binding domain in a cell-autonomous and reversible manner. Moreover, unesterified DHA increases spine formation and excitatory synaptic transmission in vivoin an Rxra-dependent fashion. RxracKO mice generally behave normally but show deficits in behavior tasks associated with social memory. Together, these results demonstrate that unesterified DHA signals through RXRA to regulate spinogenesis and functional synapse formation, providing insight into the mechanism through which DHA promotes brain development and cognitive function.