In this longitudinal study we investigate the influence of childhood disadvantage on midlife hypothalamic-pituitary-adrenal (HPA) axis regulation. Two mechanisms by which early life stress may affect later pathophysiology are through its influence on cognitive functioning or later socioeconomic (SES) disadvantage. We predicted that individual differences in young adult cognitive ability and midlife SES would mediate the influence of childhood disadvantage on midlife cortisol. On each of three nonconsecutive days, participants provided five salivary cortisol samples corresponding to their diurnal rhythm (N=727 men; mean age 55, SD=2.6). We calculated three measures of cortisol regulation (area-under-the curve cortisol reflecting total daytime cortisol output; cortisol-awakening-response; and wake-to-bed slope), averaging scores for each measure across multiple days. Childhood disadvantage combined four dichotomous indicators used previously by Rutter (1985): father low SES; mother education less than 12th grade; major family disruption/separation before age 18; and large family size (more than 5 siblings). The two mediators were a measure of general cognitive ability assessed at age 20 and highest achieved midlife SES. Men from more disadvantaged childhoods were significantly more likely to have dysregulated cortisol at midlife, with higher daytime cortisol levels decades after their childhood experience. Effects of childhood disadvantage were both direct and indirect. Cognitive ability and adult SES, however, only partially mediated the associations between early life stress and midlife cortisol. Specific indirect effects accounted for 33.8% of the total effect of childhood disadvantage [β=0.12 (0.05; 0.18)] on total daytime cortisol. Associations remained significant after accounting for ethnicity, smoking status, and self-reported depressive symptoms.