EFA6A is a guanine nucleotide exchange factor for ADP ribosylation factor 6 (Arf6), a small GTPase involved in membrane trafficking and actin cytoskeleton remodeling. While EFA6A-Arf6 signaling has been shown to regulate dendritic spine formation and maintenance in cultured neurons, its role in higher brain functions remains unclear in vivo. Here, we generated mice lacking two EFA6A splicing isoforms, EFA6A and EFA6As, to examine their role in regulating spine morphology and hippocampus-dependent learning and memory. The loss of EFA6A and EFA6As caused reduced dendritic spine density in developing CA1 pyramidal neurons, whereas dendritic spines aberrantly increased in adults. Furthermore, the mutant mice also showed impaired maintenance of long-term potentiation (LTP) at Schaffer collateral-CA1 synapses in the hippocampus and memory retention in the passive avoidance test. These findings provide the first in vivo evidence that the EFA6A isoforms, EFA6A and EFA6As, collectively regulate spine formation bidirectionally in a developmental stage-dependent manner, which is likely to underlie hippocampal synaptic plasticity and memory formation.