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Abstract Background In changing northern ecosystems, understanding the mechanisms of transmission of zoonotic pathogens, including the coccidian parasite Toxoplasma gondii, is essential to protect the health of vulnerable animals and humans. As high-level predators and scavengers, foxes represent a potentially sensitive indicator of the circulation of T. gondii in environments where humans co-exist. The objectives of our research were to compare serological and molecular assays to detect T. gondii, generate baseline data on T. gondii antibody and tissue prevalence in foxes in northern Canada, and compare regional seroprevalence in foxes with that in people from recently published surveys across northern Canada. Methods Fox carcasses (Vulpes vulpes/Vulpes lagopus, n = 749) were collected by local trappers from the eastern (Labrador and Québec) and western Canadian Arctic (northern Manitoba, Nunavut, and the Northwest Territories) during the winters of 2015–2019. Antibodies in heart fluid were detected using a commercial enzyme-linked immunosorbent assay. Toxoplasma gondii DNA was detected in hearts and brains using a magnetic capture DNA extraction and real-time PCR assay. Results Antibodies against T. gondii and DNA were detected in 36% and 27% of foxes, respectively. Detection of antibodies was higher in older (64%) compared to younger foxes (22%). More males (36%) than females (31%) were positive for antibodies to T. gondii. Tissue prevalence in foxes from western Nunavik (51%) was higher than in eastern Nunavik (19%). At the Canadian scale, T. gondii exposure was lower in western Inuit regions (13%) compared to eastern Inuit regions (39%), possibly because of regional differences in fox diet and/or environment. Exposure to T. gondii decreased at higher latitude and in foxes having moderate to little fat. Higher mean infection intensity was observed in Arctic foxes compared to red foxes. Fox and human seroprevalence showed similar trends across Inuit regions of Canada, but were less correlated in the eastern sub-Arctic, which may reflect regional differences in human dietary preferences. Conclusions Our study sheds new light on the current status of T. gondii in foxes in northern Canada and shows that foxes serve as a good sentinel species for environmental circulation and, in some regions, human exposure to this parasite in the Arctic. Graphical Abstract