Abstract Objectives Increased cholesterol levels were found to be associated with diabetes mellitus type II (DM2). The cholesterol is metabolized by cytochrome 7A1 (CYP7A1) and transported in the intestine by ATP-binding cassette G8 (ABCG8). Genetic variants in CYP7A1 and ABCG8 genes can affect the cholesterol levels. The aim of this study is to compare the frequency of CYP7A1 rs3808607 and ABCG8 rs11887534 and rs4148217 genotypes between healthy and DM2 subjects from Jordanian population. Methods A total of 117 DM2 patients and 100 healthy controls, of Jordanian Arabic origin, were genotyped for CYP7A1 rs3808607 and ABCG8 rs11887534 and rs4148217 genetic variants using polymerase chain reaction (PCR) followed by restriction fragment length polymorphism technique. Results The study showed that homozygosity of rs3808607 (A-204C) genotype in CYP7A1 was significantly higher in DM2 patients (ANOVA, p<0.05) with an odd ratio of 2.66, but rs11887534 (G55C) and rs4148217 (C1199A) genetic polymorphisms in ABCG8 were found in comparable frequencies in both healthy and DM2 subjects. Conclusions The results of this study indicate that CYP7A1 rs3808607 genetic polymorphism is associated with DM2. Further clinical studies are required to confirm this finding among DM2 patients of Jordanian origin.