Background: Gestational diabetes mellitus (GDM) is one of the prediabetes conditions in which high blood sugar levels and body weight increase during pregnancy. The underlying molecular and biochemical mechanisms of GDM are poorly defined. Introduction: Aromatase enzyme activity is responsible for the conversion of androgens to estrogens and has a share in the regulation of body fat distribution and liver receptor homolog-1 (LRH-1), which plays a critical role in cholesterol transport, acid homeostasis, and steroidogenesis in GDM patients. This study aims to determine the levels of aromatase enzyme and LRH-1 in GDM patients and to investigate the relationship between the levels of aromatase enzyme and LRH-1 and the levels of insulin, HbA 1c and total cholesterol. Method: This prospective cross-sectional study was conducted over eleven months (September 2020 to July 2021). The study population was selected at Harran University Teaching and Research Hospital. The study included 32 GDM patients and 32 healthy pregnants. The automated assay measured serum fasting blood glucose, HbA1c, and insulin levels (AVIDA 1800 Chemistry System; Siemens). Aromatase enzyme activity and LRH-1 levels were determined by using a commercial enzyme-linked immunosorbent assay (ELISA) kit. Result: Aromatase activity decreased in GDM patients while LRH1 increased. Significant differences in means levels of fasting blood glucose (p=0.11), insulin (p= 0.001) and HbA1c (p= 0.001) between the patients and control groups. There was a significant negative correlation between the levels of aromatase and insulin (r=-370, p =0.037). In addition, a positive significant correlation coefficient (r=0.645, p=0.001) was found between HbA1c and total cholesterol among the patients' group. Conclusion: Our findings indicate that there is a negative relationship between aromatase activity and insulin levels. Aromatase and LRH 1 may play a role in the pathogenesis of GDM, and the use of LRH-1 agonists in treating the disease may be considered an alternative treatment in the future. However, additional studies are required to reveal the possible functions of these two proteins in GDM with their mechanisms.