As humans continue to alter natural habitats, many wild animals are facing novel suites of environmental stimuli. These changes, including increased human–wildlife interactions, may exert sublethal impacts on wildlife such as alterations in stress physiology and behavior. California ground squirrels (Otospermophilus beecheyi) occur in human-modified as well as more pristine environments, where they face a variety of anthropogenic and naturally occurring threats. This makes this species a valuable model for examining the effects of diverse challenges on the physiology and behavior of free-living mammals. To explore potential sublethal effects of habitat modification on O. beecheyi, we compared body masses, behaviors, and fecal glucocorticoid metabolite (FGM) levels for free-living squirrels in human-disturbed versus undisturbed habitats. Prior to these analyses, we validated the use of FGMs in this species by exposing captive O. beecheyi to pharmacological and handling challenges; both challenges produced significant increases in FGMs in the study animals. While FGM responses were repeatable within captive individuals, responses by free-living animals were more variable, perhaps reflecting a greater range of life-history traits and environmental conditions within natural populations of squirrels. Animals from our human-disturbed study site had significantly higher FGMs, significantly lower body masses, and were significantly less behaviorally reactive to humans than those from our more pristine study site. Thus, despite frequent exposure of California ground squirrels to human impacts, anthropogenic stressors appear to influence stress physiology and other phenotypic traits in this species. These findings suggest that even human-tolerant mammalian species may experience important sublethal consequences due to human modifications of natural habitats.