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 vivo via retinoid X receptor α (Rxra) signaling. Using Rxra conditional knockout (cKO) mice and virus-mediated transient gene expression, we show that endogenous Rxra plays 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 vivo in an Rxra-dependent fashion. Rxra cKO 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. Display omitted •Retinoid X receptor Rxra regulates spinogenesis and excitatory synaptic transmission•RXRA effect is reversible, cell autonomous, and postsynaptic and requires DNA-binding domain•Rxra contributes to sensory experience-induced expression of immediate early genes•Unesterified DHA promotes spinogenesis and synaptic transmission via RXRA Cao et al. measure spinogenesis and synaptic transmission in Rxra cKO mice following injection of unesterified docosahexaenoic acid (DHA). They demonstrate that unesterified DHA signals via RXRA to regulate spinogenesis and functional synapse formation, providing insight into the mechanism through which DHA promotes brain development and cognitive function.