Interactions with humans influence the behavior and physiology of other animals, and the response can vary with sex and familiarity. Dogs in animal shelters face challenging conditions and although contact with humans typically reduces stress and behaviors associated with stress, evidence indicates that shelter dogs react differently to unfamiliar men and women. Given that some aspects of canine scent-marking behavior change under fearful conditions, we examined whether sex of an unfamiliar walker would influence scent-marking behavior of 100 shelter dogs during leash walks. Male dogs urinated at higher rates when walked by unfamiliar women than when walked by unfamiliar men; female dogs urinated at similar rates when walked by unfamiliar women and unfamiliar men. Sex of walker influenced urinary posture in male dogs, but not in female dogs. Both male and female dogs were more likely to defecate when walked by unfamiliar women than by unfamiliar men. Based on our findings that shelter dogs behave differently in the presence of unfamiliar men and women, we suggest that researchers conducting behavioral studies of dogs record, consider in analyses, and report the sex of observers and handlers as standard practice. We also recommend recording the sex of shelter staff present at behavioral evaluations because the results of these evaluations can impact dog welfare.