Interleukin 35 (IL-35) functions in an anti-inflammatory fashion by inhibiting T-cell proliferation, whereas CD4(+) T cells play an important role in cellular immunity. In a hepatitis B virus (HBV) infection, the viral proteins stimulate the immune system to generate antiviral molecules, which correlate to HBV DNA load. We investigated the impact of HBV DNA load on the expression of IL-35 mRNA in CD4(+) T cells, and the expression of IL-35 cytokine in serum of the patients with chronic HBV infection. Here we report that the frequency of circulating CD4(+) T cells correlates with the HBV DNA load in the serum of chronic hepatitis B (CHB) patients. An increased number of CD4(+) T cells were found in those patients with higher levels of HBV DNA. Regulatory T cells (T regs) also showed this trend, but circulating cytotoxic lymphocytes (CTLs) showed a negative correlation with serum HBV DNA load. In addition, significantly more IL-35 mRNA was found in the CD4(+) T cells of CHB patients, compared to healthy controls. Patients in the high viral load group showed increased levels of IL-35 mRNA, compared with those in the low viral load group. The level of IL-35 cytokine in the serum of CHB patients was significantly higher than in the healthy controls and in those infected with HBV, the patients with a higher viral load had more serum IL-35 cytokines, compared to those with a lower viral load. Our study suggests that increased serum IL-35 could be directly related to increased levels of IL-35 mRNA in CD4(+) T cells and HBV DNA load in CHB patients. The possible role of IL-35 as an immune regulator in chronic HBV infection should be investigated further.