Hepatitis C virus (HCV) is a leading cause of chronic liver disease in the world, making this disease a significant cause of morbidity and mortality. This study aimed to evaluate the interrelation between the vitamin D serum level and the functional polymorphism of 1-alpha hydroxylase (CYP27B1) in the response of chronic HCV patients to treatment. The study was conducted in the Medical Research Centre, Faculty of Medicine, Ain Shams University, and included thirty patients diagnosed as chronic hepatitis C who were treated with pegylated interferon (PEG-IFN) and ribavirin (RBV) combination therapy. Twenty healthy subjects were also included as a control group. These patients were divided into two groups; responders (10 patients) and non-responders (20 patients). The blood samples were taken for assay of vitamin D level and CYP27B1 polymorphism. The results revealed that chronic HCV patients were suffering from deficiency of both 25(OH)-vitamin D and 1,25 dihydroxyvitamin D (1,25(OH) 2 D), but with respect to CYP27B1 genetic polymorphism, no specific genotype for chronic HCV patient. However, responders with genotype AA of the CYP27B1 had higher serum concentrations of 1,25(OH) 2 D than the responders with genotypes AC and CC. In conclusion, HCV-infected patients carrying homozygous AA of the CYP27B1 have higher chances to respond to (PEG-IFN)-based treatment.