Gestational diabetes mellitus (GDM) is a pregnancy-related metabolic disorder associated with short-term and long-term adverse health outcomes, but its pathogenesis has not been clearly elucidated. Investigations of the dynamic changes in metabolomic markers in different trimesters may reveal the underlying pathophysiology of GDM progression. Therefore, in the present study, we analysed the metabolic profiles of 75 women with GDM and 75 women with normal glucose tolerance throughout the three trimesters. We found that the variation trends of 38 metabolites were significantly changed during GDM development. Specifically, longitudinal analyses revealed that cysteine (Cys) levels significantly decreased over the course of GDM progression. Further study showed that Cys alleviated GDM in female mice at gestational day 14.5, possibly by inhibiting phosphoenolpyruvate carboxykinase to suppress hepatic gluconeogenesis. Taken together, these findings suggest that the Cys metabolism pathway might play a crucial role in GDM and Cys supplementation represents a potential new treatment strategy for GDM patients.