We examined whether the effect of the apolipoprotein E (APOE) genotype on functional brain connectivity is modulated by gender in healthy older human adults. Our results confirm significantly decreased connectivity in the default mode network in healthy older APOE ε4 carriers compared with ε3 homozygotes. More important, further testing revealed a significant interaction between APOE genotype and gender in the precuneus, a major default mode hub. Female ε4 carriers showed significantly reduced default mode connectivity compared with either female ε3 homozygotes or male ε4 carriers, whereas male ε4 carriers differed minimally from male ε3 homozygotes. An additional analysis in an independent sample of healthy elderly using an independent marker of Alzheimer's disease, i.e., spinal fluid levels of tau, provided corresponding evidence for this gender-by-APOE interaction. Together, these results converge with previous work showing a higher prevalence of the ε4 allele among women with Alzheimer's disease and, critically, demonstrate that this interaction between APOE genotype and gender is detectable in the preclinical period.