Following precipitous population declines as a result of intensive hunting and 20th century predator-control programmes, hybridization of the Critically Endangered red wolf Canis rufus with coyotes Canis latrans posed a significant challenge for red wolf recovery efforts. Anthropogenic mortality and hybridization continue to pose challenges; the increasing number of wolf deaths caused by humans has limited wolf population growth, facilitated the encroachment of coyotes into eastern North Carolina, and affected the formation and disbandment of breeding pairs. We assessed the effects of anthropogenic mortality on Canis breeding units during a 22-year period (1991–2013). Our results show that deaths caused by people accounted for 40.6% of breeding pair disbandment, and gunshots were the primary cause of mortality. Red wolves replaced congeneric breeding pairs > 75% of the time when pairs disbanded under natural conditions or as a result of management actions. Since the mid 2000s anthropogenic mortality has caused annual preservation rates of red wolf breeding pairs to decline by 34%, and replacement of Canis breeders by red wolves to decline by 30%. Our results demonstrate that human-caused mortality, specifically by gunshots, had a strong negative effect on the longevity of red wolf pairs, which may benefit coyotes indirectly by removing their primary competitor. Coyotes are exacerbating the decline of red wolves by pair-bonding with resident wolves whose mates have been killed.