Monitoring the hypothalamic–pituitary–adrenal (HPA) axis through determination of fecal cortisol metabolite (FCM) levels is a non-invasive method useful for understanding how handling and social conditions may affect the physiological status of zoo animals. The present study used FCM analysis to evaluate whether the HPA axis activity of a lion pride was modified by a change in social and handling conditions after the death of the dominant male. Five African lions (Panthera leo bleyenberghi), two males and three females, were included in the study. Fecal samples were collected before and after the death of the dominant male. To avoid cohabitation conflicts between males before the dominant male died, subgroups were established and subjected to weekly changes between indoor and outdoor facilities. After the death of the dominant male, these management dynamics ceased, and the remaining four lions were kept together outdoors. Significant lower group FCM concentrations (p < 0.001) were detected after the decease of the dominant male, probably associated with a decrease in daily handling, together with a more stable social environment. Overall, the present study indicates the effect of different management scenarios on the HPA axis activity and differentiated physiological responses to the same situation between individuals.