Subtle alterations in dendritic spine morphology can induce marked effects on connectivity patterns of neuronal circuits and subsequent cognitive behavior. Past studies of rodent and nonhuman primate aging revealed reductions in spine density with concomitant alterations in spine morphology among pyramidal neurons in the prefrontal cortex. In this report, we visualized and digitally reconstructed the three-dimensional morphology of dendritic spines from the dorsolateral prefrontal cortex in cognitively normal individuals aged 40–94 years. Linear models defined relationships between spines and age, Mini–Mental State Examination, apolipoprotein E (APOE) ε4 allele status, and Alzheimer's disease (AD) pathology. Similar to findings in other mammals, spine density correlated negatively with human aging. Reduced spine head diameter associated with higher Mini–Mental State Examination scores. Individuals harboring an APOE ε4 allele displayed greater numbers of dendritic filopodia and structural alterations in thin spines. The presence of AD pathology correlated with increased spine length, reduced thin spine head diameter, and increased filopodia density. Our study reveals how spine morphology in the prefrontal cortex changes in human aging and highlights key structural alterations in selective spine populations that may promote cognitively normal function despite harboring the APOE ε4 allele or AD pathology.