Antibiotic resistome has emerged as a global threat to public health. However, gestational antibiotic resistome and potential link with adverse pregnancy outcomes remains poorly understood. Our study reports for the first time an association between gut antibiotic resistome during early pregnancy and the risk of gestational diabetes mellitus (GDM) based on a prospective nested case–control cohort including 120 cases and 120 matched controls. A total of 214 antibiotic resistance gene (ARG) subtypes belonging to 17 ARG types were identified in > 10% fecal samples collected during each trimester. The data revealed dynamic profiles of gut antibiotic resistome through pregnancy, and significant positive associations between selected features (i.e., ARG abundances and a GDM−ARG score which is a new feature characterizing the association between ARGs and GDM) of gut antibiotic resistome during early pregnancy and GDM risk as well as selected endogenous metabolites. The findings demonstrate ubiquitous presence of ARGs in pregnant women and suggest it could constitute an important risk factor for the development of GDM.