Synaptic loss is a symptom of Alzheimer's disease (AD) that is associated with the onset of cognitive decline and the loss of executive function. The strongest genetic risk factor for AD is the APOE4 allele, which results in both a greater risk of developing AD as well as an earlier age of onset of AD. Dendritic spines, the anatomical substrate of the excitatory synapse, are reduced in the cortex of humanized APOE4 mice but the reason for this synaptic decline is unknown. Calcineurin, a calcium/calmodulin dependent phosphatase, is a mediator of dendritic spine retraction. We used humanized APOE mice to examine how APOE genotype altered calcineurin activity and found that APOE4 mice have 35% higher cortical calcineurin activity compared with APOE3 mice. This occurred in the absence of any increase in calcineurin protein levels or mRNA expression. The elevation in calcineurin was associated with 10% fewer dendritic spine number in layer II/III of the cortex. Treatment with the calcineurin inhibitor FK506 reduced calcineurin activity by 64% and resulted in normalization of dendritic spine numbers in APOE4 mice. In conclusion, we found that the APOE4 gene in mice was associated with elevated calcineurin activity and fewer dendritic spine numbers compared with APOE3 mice. Importantly, calcineurin in APOE4 remained sensitive to pharmacological inhibition and spine density can be rescued by treatment with FK506.