Background/Aim: Gestational diabetes (GDM) is a complex metabolic condition associated with hyperpglycemia that is diagnosed in an oral glucose tolerance test (OGTT) during pregnancy. For a deeper understanding of the pathology of the disease, further investigations during pregnancy are required, ideally under metabolic challenging conditions. Methods: We performed targeted metabolomics in a group of 24 well-matched women during an oral glucose tolerance test (OGTT). 231 plasma metabolites were profiled and compared to conventional clinical diagnostics. Results: A pattern of 8 metabolites differed between GDM and healthy controls as early as 30min in an OGTT (AUC 0.9770.008), and an increase in acylcarnitine C18:0, decreased concentrations of diacyl phosphatidylcholines (PC aa) C34:4, PC aa C36:4, PC aa C38:5, Lyso PC C20:4 and arachidonic acid were associated with insulin resistance. Conclusion: Our data suggest an additional value of metabolite pattern in the diagnosis of GDM and describe altered pathways that might be subjected to a more precise diagnosis and individualized therapy.