In this study, we sought to determine whether the affective and physiological experience of primiparous, or first-time, motherhood is distinct from multiparous motherhood, how the child’s level of inhibited temperament impacts it, and if such a temperament results in overprotective parenting behaviors. A total of 117 mothers and their 24-month-old toddlers participated in novelty tasks designed to elicit parenting behaviors and toddler’s typical fear reactions. Mothers also completed a battery of questionnaires. Results suggest that primiparous mothers experienced more worry, which was associated with increased overprotective parenting behaviors. Primiparous mothers also demonstrated greater physiological (i.e., cortisol) reactivity while watching their first-born children interact with novel stimuli, but how this related to overprotective parenting was dependent on the child’s level of inhibition. Specifically, primiparous mothers displayed more cortisol reactivity with their uninhibited toddlers, which indirectly linked parity to less overprotective parenting behaviors. Primiparous mothers of highly inhibited toddlers displayed greater overprotective parenting behaviors, independent of maternal cortisol reactivity. The results indicate that the transition to motherhood is a unique experience associated with greater worry and physiological reactivity and is meaningfully influenced by the toddler’s temperament. Distinctions in both observed and self-reported overprotective parenting are evident through considering the dynamic interaction of these various aspects.