The current study is one of the first prospective examinations of longitudinal associations between observed father caregiving behaviors and child cortisol reactivity and regulation in response to emotional arousal. Observations of father and mother caregiving behaviors and child cortisol levels in response to challenges at 7 months and 24 months of child age were collected. Analyses were based on a subsample of children from the Family Life Project who lived with both their biological mothers and fathers and for whom there was at least partial cortisol data (7 months: n = 717; 24 months: n = 579). At the challenge conducted at 7 months of child age, 49.0% of the sample were girls; racial composition of the sample was 25.8% African American and 74.2% European American. At the challenge conducted at 24 months of child age 49.9% of the sample were girls; racial composition was 24.7% African American and 75.3% European American. We conducted analyses across assessment points simultaneously using mixed linear modeling for repeated measures data to test for differential effects of fathering across infancy and toddlerhood. Concurrent measures of father negativity were positively associated with greater increases in child cortisol levels in response to emotion challenge at 7 months (p = 0.01) and with higher overall levels of cortisol at 24 months (p less than 0.001). However, there was no evidence that father caregiving during infancy independently predicted later cortisol activity during toddlerhood. (Contains 2 figures and 4 tables.)