Gestational diabetes mellitus (GDM) is a global health issue, whereby pregnant women are afflicted with carbohydrate intolerance with first onset during pregnancy. GDM is characterized by maternal peripheral insulin resistance, thought to be driven by low-grade maternal inflammation. Nobiletin, a polymethoxylated flavonoid, possesses potent glucose-sensitizing and anti-inflammatory properties; however, its effects in GDM have not been assessed. The present study aimed to determine the effects of nobiletin on glucose metabolism and inflammation associated with GDM in both in vitro human tissues and an in vivo animal model of GDM. In vitro, treatment with nobiletin significantly improved TNF-impaired glucose uptake in human skeletal muscle, and suppressed mRNA expression and protein secretion of pro-inflammatory cytokines and chemokines in human placenta and visceral adipose tissue (VAT). Mechanistically, nobiletin significantly inhibited Akt and Erk activation in placenta, and NF-kappa B activation in VAT. In vivo, GDM mice treated with 50 mg/kg nobiletin daily via oral gavage from gestational day (gd) 1-17 or via i.p. injections from gd 10-17 significantly improved glucose tolerance. Pregnant GDM mice treated with nobiletin from either gd 1-17 or gd 10-17 exhibited significantly suppressed mRNA expression of pro-inflammatory cytokines and chemokines in placenta, VAT and subcutaneous adipose tissue (SAT). Using a quantitative mass spectrometry approach, we identified differentially abundant proteins associated with the effect of nobiletin in vivo. Together, these studies demonstrate that nobiletin improves glucose metabolism and reduces inflammation associated with GDM and may be a novel therapeutic for the prevention of GDM.