Variations in maternal behavior are associated with differences in estrogen receptor (ER)-α expression in the medial preoptic area (MPOA) and are transmitted across generations such that, as adults, the female offspring of mothers that exhibit increased pup licking/grooming (LG) over the first week postpartum (i.e. high LG mothers) show increased ERα expression in the MPOA and are themselves high LG mothers. In the present studies, cross-fostering confirmed an association between maternal care and ERα expression in the MPOA; the biological offspring of low LG mothers fostered at birth to high LG dams show increased ERα expression in the MPOA. Cross-fostering the biological offspring of high LG mothers to low LG dams produces the opposite effect. We examined whether the maternal programing of ERα expression is associated with differences in methylation of the relevant ERα promoter. Levels of cytosine methylation across the ERα1b promoter were significantly elevated in the adult offspring of low, compared with high, LG mothers. Differentially methylated regions included a signal transducer and activator of transcription (Stat)5 binding site and the results of chromatin immunoprecipitation assays revealed decreased Stat5b binding to the ERα1b promoter in the adult offspring of low, compared with high, LG mothers. Finally, we found increased Stat5b levels in the MPOA of neonates reared by high, compared with low, LG mothers. These findings suggest that maternal care is associated with cytosine methylation of the ERα1b promoter, providing a potential mechanism for the programming of individual differences in ERα expression and maternal behavior in the female offspring.