Objective Although gestational diabetes mellitus (GDM) is associated with an increased risk of type 2 diabetes mellitus (T2DM) compared to normoglycemic pregnancies, the biochemical pathways underlying the progression of GDM to T2DM are not fully elucidated. The purpose of this exploratory study was to utilize metabolomics with an oral glucose tolerance test (OGTT) to examine the amino acid response in women with prior GDM to determine if a relationship between these metabolites and established risk factors for T2DM exists. Materials/Methods Thirty-eight non-pregnant women without diabetes but with prior GDM within the previous 3 years were recruited from a community-based population. A 75 g-OGTT was administered; fasting and 2-hr plasma samples were obtained. Metabolite profiles of 23 amino acids or amino acid derivatives were measured with gas chromatography-mass spectrometry. Measures of insulin resistance were derived from the OGTT and risk factors for T2DM were obtained by self-report. Results Twenty-two metabolite levels decreased significantly in response to the OGTT (p<0.05). The clinical covariates most powerfully associated with metabolite level changes included race, body mass index (BMI), and duration of prior breastfeeding, (mean ± SD of standardized β-coefficients, β = −0.38 ± 0.05, 0.25 ± 0.08, and 0.44 ± 0.03, respectively, all p<0.05). Notably, a prior history of breastfeeding was associated with the greatest number of metabolite changes. Conclusions Greater change in metabolite levels after a glucose challenge was significantly associated with a longer duration of breastfeeding and higher BMI. Further exploration of these preliminary observations and closer examination of the specific pathways implicated are warranted.