Objective To investigate the mechanism by which miR-335-5P regulates the transforming factor-β (TGF-β) pathway in promoting islet β cell secretion and enhancing insulin resistance in gestational diabetes mellitus (GDM) mice. Methods Specific pathogen free grade Balb / c mice were reared in cages at a female to male ratio of 2 ∶1. Ten normal pregnant mice were assigned to the blank group, and 48 mice were prepared as GDM model mice and divided into six groups for different drug treatments—groups A, B, C, D, E and F—with eight mice in each group. After drug treatment, fasting glucose (FBG) and fasting insulin (FIRI) were detected by the oral glucose tolerance test (OGTT), and the insulin resistance index (IRI) was calculated. The hyperinsulinemic-euglycemic clamp was used to determine the glucose infusion rate (GIR) and to estimate pancreatic islet β cell function. The levels of miR-335-5P and key islet β cell TGF-β pathway factor mRNAs and proteins were determined by RT-qPCR, western blot and ELISA analysis. Results After drug treatment, FBG, FIRI and IRI in groups D and F were decreased and were lower than respective values of groups C and E (P<0. 05). GIR, first-phase insulin and maximum insulin in groups D and F were increased and were higher than respective values of groups C and E (P<0. 05). The levels of miR-335-5P, TGF-β and c-Myc in groups D and F were lower than those of groups A, B, C and E (P<0. 05). When miR-335-5P expression was detected in islet β cells, TGF-β and c-Myc were expressed at different levels. TGF-β was expressed in the cytoplasm of islet β cells, and the expression of TGF-β in groups D and F was higher than that in groups A, B, C and E (P<0. 05). Conclusions miR-335-5P can up- regulate c-Myc expression in GDM mice, activate the TGF-β pathway, enhance insulin resistance and inhibit islet β-cell secretion.