The P85 regulatory subunit protein and gene expression and P110 catalylic subunit activity of phosphatidylinositol 3-kinase (PI-3K) were investigated in adipose tissue of patients with gestational diabetes mellitus (GDM) in order to explore the molecular mechanisms of insulin resistance (IR) of GDM. Samples from patients with GDM (n=50), and controls (n=50) were collected. Fasting insulin (FIN) was determined by radioimmunoassay. Fasting plasma glucose (FPG) was measured by oxidase assay. Western blot technique was used to detect the levels of PI-3K P85 subunit in adipose tissues of patients with GDM. The mRNA expression of PI-3K P85 subunit was detected by reverse transcription polymerase chain reaction (RT-PCR) method in the adipose tissue. PI-3K activity was examined by immunoprecipitation, thin-layer chromatography and gamma scintillation counting. The results were analyzed statistically. It was found that the levels of FPG, FIN and HOMA-IR in GDM group were significantly higher than those in control group (all P < 0.01). There was no significant difference in the protein and gene expression of PI-3K P85 subunit between GDM group and control group (P > 0.05). PI-3K activity was significantly decreased to 82.89% in GDM group as compared with control group (P < 0.01) and negatively correlated with HOMA-IR (r=-0.75, P < 0.01). It was concluded that PI-3K in GDM patients may be involved in the insulin signaling pathway, resulting in IR of GDM.