Costimulatory signals are critical for antiviral immunity. The aim of this study was to evaluate the change of costimulatory molecule CD28 on circulating CD8+ T cells in chronic hepatitis B patients (CHB). Seventy CHB patients and fifty-six healthy controls were included, and forty-eight CHB patients were recruited for 52 weeks of longitudinal investigation. The proportions of circulating CD8+CD28+ and CD8+CD28- subpopulations were determined by flow cytometry, and the CD8+CD28+/CD8+CD28- T cells ratio was calculated. Compared with the subpopulation in healthy controls, high proportions of CD8+CD28- subpopulation were observed in CHB patients. Similarly, the CD8+CD28+/CD8+CD28- T cells ratio was significantly decreased in CHB patients compared with healthy controls and correlated significantly with hepatitis B virus (HBV) loads. High proportions of CD8+CD28- subpopulation and low CD8+CD28+/CD8+CD28- T cells ratio were observed in hepatitis B e antigen- (HBeAg-) positive individuals as compared with that in HBeAg-negative subjects. A significant decrease in CD8+CD28- subpopulation, increase in CD8+CD28+ subpopulation, and CD8+CD28+/CD8+CD28- T cells ratio were seen in those patients who received efficient antiviral therapy. Thus, aberrant CD28 expression on circulating CD8+ T cells and the CD8+CD28+/CD8+CD28- T cells ratio reflect the dysregulation of T cell activation and are related to the pathogenesis of chronic HBV infection.