African elephants (Loxodonta africana) are intensively managed in southern Africa and are routinely translocated between reserves. Domesticated elephants are used for elephant-back safaris and interactions with guests. Understanding how elephants respond to such activities is critical because of welfare issues associated with both humans and elephants. We investigated the stress response (i.e., fecal glucocorticoid metabolite secretion [FGM]) of working elephants in Letsatsing Game Reserve, South Africa, over 1 year to evaluate their response to transportation and ecotourism activities. We used free-ranging elephants in adjacent Pilanesburg National Park as controls. Fecal glucocorticoid metabolites were greatest prior to and during translocation and declined over the year. Within 1–2 months of transportation, FGM levels in working elephants became indistinguishable from those in wild elephants. Fecal glucocorticoid metabolite levels were higher during human interaction days than days without interaction. The highest observed FGM levels were associated with transportation and episodic loud noises. Transportation is a stressful activity for elephants, and ≥3 months should be provided to translocated elephants to acclimate to their new surroundings. Although stress levels of elephants increased slightly when interacting with humans in the contexts we studied, evaluating interactions under a wider range of contexts is necessary to minimize danger to elephants and humans.