Children and families from socioeconomically marginalized background experience high levels of stress, especially persistent chronic stress, due to unstable housing, employment, and food insecurity. Although consistent evidence supports a stress-obesity connection, little research has examined the potential moderation role of stress in childhood obesity interventions. Therefore, this study aimed to explore how chronic stress (hair cortisol) moderated the effects of a healthy lifestyle intervention on improving behavioral and anthropometric outcomes among 95 socioeconomically marginalized parent-child dyads. Data were collected in a cluster randomized controlled trial with 10 Head Start childcare centers being randomized into intervention and control. The child sample (3–5 years old) included 57.9 % female, 12.6 % Hispanic, and 40.0 % Black. For the parents, 91.6 % were female, 8.4 % were Hispanic, 36.8 % were Black, and 56.8 % were single. Parent baseline hair cortisol significantly moderated the intervention effects on child fruit intake (B = -1.56, p = .030) and parent nutrition self-efficacy (B = 1.49, p = .027). Specifically, higher parent hair cortisol lowered the increases in child fruit intake but improved the increases in parent nutrition self-efficacy in the intervention group compared to control group. Child higher baseline hair cortisol was significantly associated with the decreases in child fruit intake (B = -0.60, p = .025). Child baseline hair cortisol significantly moderated the intervention effects on parent physical activity (PA) self-efficacy (B = -1.04, p = .033) and PA parental support (B = -0.50, p = .016), with higher child hair cortisol decreasing the improvement on these two outcomes in the intervention group compared to control group. Results from this study shed lights on the moderation role of chronic stress on impacting healthy lifestyle intervention effects. Although needing further investigation, the adverse effects of chronic stress on intervention outcomes should be considered when developing healthy lifestyle interventions for preschoolers and their families.