Diabetes is a metabolic disease with the characteristic of high blood glucose (hyperglycemia). In our previous study, we found that nigelladines A–C (compounds A–C), three norditerpenoid alkaloids from the seeds of Nigella glandulifera Freyn (Ranunculaceae) exhibited protein of tyrosine phosphatase 1B (PTP1B) inhibitory activity in vitro. In the present study, we further investigated their anti-diabetes activities in L6 moytubes and illuminated the mechanisms of action of compounds A–C. Several parameters of glucose metabolism such as glucose consumption, glycogen content and hexokinase activity were increased by compounds A–C. The results suggested that compounds A–C improved glucose metabolism through promoting synthesis of glycogen. Expression of PTP1B protein was inhibited by compounds A–C in L6 moytubes. PI3K-dependent Akt phosphorylation was found to be activated by compounds A-C and completely blocked by wortmannin (a PI3K inhibitor). Moreover, the insulin-mediated induction of insulin receptor substrate-1 (IRS-1) and glycogen synthase kinase-3β (GSK-3β) were also suppressed by wortmannin. Western blot results indicated that compounds A–C-induced IRS-1/Akt activation was likely a consequence of PTP1B inhibition. Compounds A–C promoted glycogen synthesis through Akt-mediated GSK3 phosphorylation. Therefore, activation of PI3K/Akt insulin signaling pathway and suppression of PTP1B is the molecular mechanism that contributes to the anti-diabetic effect of compounds A–C in cellular models. The three alkaloids potentially serve as lead compounds for the development of antidiabetic drugs.