The present study examined the concurrent and prospective, longitudinal effects of childhood negative emotionality and self-regulation on allostatic load (AL), a physiological indicator of chronic stress. We hypothesized that negative emotionality in combination with poor self-regulation would predict elevated AL. Mothers reported on children's emotionality (N = 239, 116 males) at age 9, and self-regulation was measured at age 9 using a standard behavioural index of delay of gratification. AL was measured at ages 9 and 17. The AL measure captured hypothalamic pituitary adrenal axis, sympathetic adrenal medullary system, cardiovascular, and metabolic activity. Our results confirmed that negative emotionality prospectively predicted increases in AL, but only in children with low levels of self-regulation. The findings suggest that the link between negative emotionality and stress physiology is moderated by self-regulation and contribute to understanding the role of negative emotionality in physiological functioning throughout childhood and adolescence.