Liver enzymes are independent predictors of type 2 diabetes. Although liver fat content correlates with features of insulin resistance, a risk factor for developing gestational diabetes mellitus (GDM), the relationship between liver enzymes and GDM is unclear. The objective of this study was to assess whether pregravid liver enzyme levels are associated with subsequent risk of GDM. A nested case-control study was conducted among women who participated in the Kaiser Permanente Northern California multiphasic health checkup (1984-1996) and had a subsequent pregnancy (1984-2009). Case patients were 256 women who developed GDM. Two control subjects were selected for each case patient and matched for year of blood draw, age at examination, age at pregnancy, and number of intervening pregnancies. Being in the highest quartile versus the lowest quartile of ?-glutamyl transferase (GGT) levels was associated with a twofold increased risk of subsequent GDM (odds ratio 1.97 [95% CI 1.14-3.42]), after adjusting for race/ethnicity, prepregnancy BMI, family history of diabetes, and alcohol use. This result was attenuated after adjusting for homeostasis model assessment of insulin resistance (HOMA-IR), fasting status, and rate of gestational weight gain. There was significant interaction between GGT and HOMA-IR; the association with GGT was found among women in the highest tertile of HOMA-IR. Aspartate aminotransferase and alanine aminotransferase were not associated with increased GDM risk. Pregravid GGT level, but not alanine aminotransferase or aspartate aminotransferase level, predicted the subsequent risk of GDM. Markers of liver fat accumulation, such as GGT level, are present years before pregnancy and may help to identify women at increased risk for subsequent GDM.