Beyond bacteria, the human gastrointestinal tract is host to a vast diversity of fungi, collectively known as the gut mycobiome. Little is known of the impact of geography, ethnicity and urbanization on the gut mycobiome at a large population level. We aim to delineate the variation of human gut mycobiome and its association with host factors, environmental factors and diets. Using shotgun metagenomic sequencing, we profiled and compared the fecal mycobiome of 942 healthy individuals across different geographic regions in China (Hong Kong and Yunnan), spanning six ethnicities, Han, Zang, Bai, Hani, Dai, and Miao (including both urban and rural residents to each ethnicity). In parallel to fecal sampling, we collected subject metadata (environmental exposure, bowel habits, anthropometrics and medication), diet and clinical blood measurement results (a total of 118 variables) and investigated their impact on the gut mycobiome variation in humans. The human gut mycobiome was highly variable across populations. Urbanization-related factors had the strongest impact on gut mycobiome variation, followed by geography, dietary habit, and ethnicity. The Hong Kong population (highly urbanized) had a significantly lower fungal richness compared with Yunnan population. Saccharomyces cerevisiae was highly enriched in urban compared with rural populations, and showed significant inverse correlations with liver pathology-associated blood parameters, including aspartate transaminase (AST), alanine transaminase (ALT), gamma glutamyltransferase (GGT), and direct bilirubin. Candida dubliniensis, which was decreased in urban relative to rural populations, showed correlations with host metabolism-related parameters in blood, including a positive correlation with fasting high-density lipoprotein cholesterol (HDL-C) levels and a negative correlation with fasting glucose levels. The fungal-blood parameter correlations were highly geography- and ethnicity-specific. Food choices had differential influences on gut mycobiome and bacterial microbiome, where taxa from the same genus tended to be co-regulated by food and thereby co-bloom. Ethnicity-specific fungal signatures were associated with distinct habitual foods in each ethnic group. Our data highlight for the first time that geography, urbanization, and ethnicity, and habitual diets play an important role in shaping the gut mycobiome composition. Gut fungal configurations in combination with population characteristics (such as residing region, ethnicity, diet, lifestyle) influence host metabolism and health. Copyright © 2020 AGA Institute. Published by Elsevier Inc. All rights reserved.