Hepatitis C virus (HCV) is a blood-borne pathogen which has chronically infected people worldwide. Therefore, it is of utmost importance to design prophylactic and therapeutic vaccine in order to control HCV infection. To date, several researchers have attempted to improve the efficiency of HCV vaccine by using different adjuvants. However, a few studies have focused on the synthetic immunomodulatory drugs as adjuvants for HCV vaccine. Recently, researchers have shown that lenalidomide, which is used to treat the patients with multiple myeloma, is capable of improving the immune system factors. In this paper, two doses of lenalidomide along with pcDNA3.1 + NS3 as HCV DNA vaccine were administrated in mice models and the percentage of regulatory T cells (Treg cells) and the cells with PD-1 + expression in spleen of mice model were investigated by flow cytometry method. Additionally, activities of CTL cells and NK cells were evaluated in spleen of prophylactic and therapeutic mice models via LDH method. Results of the Treg and PD-1 analysis showed that low dose of lenalidomide along with pcDNA3.1 + NS3 can noticeably decrease the percentage of Treg cells and the cells with PD-1 + expression, while lenalidomide can significantly increase the CTL and NK activity in mice models. Also, results of the therapeutic mice model, in which SP2/0 cells- challenged mice were treated with 5 mg/kg lenalidomide in combination with pcDNA3.1 + NS3, reasonably agreed with those of the prophylactic model. Finally, it was found that lenalidomide can reduce the level of Treg cells which results in lower the cells with PD-1 + expression and subsequently higher CTL and NK cell activities. This study concluded that lenalidomide possess the characteristics of an ideal adjuvant candidate for use in combination with HCV DNA vaccine in order to promote the immune response and vaccine efficiency.