--- - |2- Abstract Multiple studies have reported different methods in treating gestational diabetes mellitus (GDM); however, the relationship between miR‐335‐5p and GDM still remains unclear. Here, this study explores the effect of miR‐335‐5p on insulin resistance and pancreatic islet β‐cell secretion via activation of the TGFβ signaling pathway by downregulating VASH1 expression in GDM mice. The GDM mouse model was established and mainly treated with miR‐335‐5p mimic, miR‐335‐5p inhibitor, si‐VASH1, and miR‐335‐5p inhibitor + si‐VASH1. Oral glucose tolerance test (OGTT) was conducted to detect fasting blood glucose (FBG) fasting insulin (FINS). The OGTT was also used to calculate a homeostasis model assessment of insulin resistance (HOMA‐IR). A hyperglycemic clamp was performed to measure the glucose infusion rate (GIR), which estimated β‐cell function. Expressions of miR‐335‐5p, VASH1, TGF‐β1, and c‐Myc in pancreatic islet β‐cells were determined by RT‐qPCR, western blot analysis, and insulin release by ELISA. The miR‐335‐5p mimic and si‐VASH1 groups showed elevated blood glucose levels, glucose area under the curve (GAUC), and HOMA‐IR, but a reduced GIR and positive expression of VASH1. Overexpression of miR‐335‐5p and inhibition of VASH1 contributed to activated TGFβ1 pathway, higher c‐Myc, and lower VASH1 expressions, in addition to downregulated insulin and insulin release levels. These findings provided evidence that miR‐335‐5p enhanced insulin resistance and suppressed pancreatic islet β‐cell secretion by inhibiting VASH1, eventually activating the TGF‐β pathway in GDM mice, which provides more clinical insight on the GDM treatment. - Journal of Cellular Physiology, EarlyView.