To investigate the correlation between the levels of microRNA-27 (miR-27) and peroxisome proliferator activated receptor-γ (PPAR-γ) of women with gestational diabetes mellitus (GDM) and their insulin resistance (IR). Methods: From May 2017 to July 2020, 131 pregnant women with GDM were selected in study group, and 135 healthy pregnant women were selected in control group during the same period. Real-time quantitative PCR (qRT-PCR) was used to detect the expression level of serum miR-27 of the women, and the level of serum PPAR-γof the women was detected by enzyme-linked immunosorbent assay (ELISA). The levels of fasting blood glucose (FBG) and fasting insulin (FINS) of the women were detected, and their homeostasis model insulin resistance index (HOMA-IR) was calculated. Pearson method was used to analyze the correlation between the levels of serum miR-27 and PPAR-γ and the levels FINS level, FBG level, and HOMA-IR value, and also to analyze the correlation between serum miR-27 level and PPAR-γlevel. Results: The levels of serum miR-27 (1.75±0.58), FINS (11.38±3.84 mIU/L), FBG (5.69±1.92 mmol/L), and HOMA-IR value (2.86±0.97) of the women in the study group were significant higher than those of the women in the control group, but the PPAR-γ level (5.47±1.89 pg/ml) of the women in the study group was significant lower (all P＜0.05). The serum miR-27 level of the women with GDM was positive correlated with their HOMA-IR value, and was negative correlated with their PPAR-γ level. The serum PPAR-γ level of the women with GDM was negative correlated with their HOMA-IR value (all P＜0.05). Conclusion: The expression of serum MiR-27 of pregnant women with GDM is up-regulated, while the level of PPAR-γ is down regulated. The expression of serum MiR-27 is negative correlated with the level of PPAR–γ， both of serum MiR-27 level and PPAR–γ level are correlated with the value of IR, so the MiR-27 and PPAR –γlevels may be the potential targets for treating GDM.