Whether hepatitis B virus (HBV) infection impairs human infertility is unclear. The present retrospective case–controlled study investigated the impact of HBV on sperm parameters, ovarian stimulation, and outcomes of in vitro fertilization (IVF) and embryo transfer. A total of 224 couples with at least one partner being HBsAg-seropositive undergoing their first IVF and embryo transfer cycle were identified, which included 77 couples with female partners being HBsAg-seropositive, 136 couples with male partners being HBsAg-seropositive, and 11 couples with both partners being HBsAg-seropositive. A total of 448 both HBsAg-seronegative couples served as controls. The percentage of normal sperm morphology was significantly lower in HBsAg-seropositive male partners than that in HBsAg-seronegative male partners (11.9 ± 9.4% vs. 19.0 ± 11.9%, P < 0.01). The duration of infertility was significantly prolonged in HBV-seropositive patients compared with HBV-seronegative patients (4.9 vs. 4.1 years, P < 0.01). Couples with female partners being HBsAg-seropositive had significantly lower top-quality embryo rate than control group (22.4% vs. 31.6%, P < 0.01). In addition, the fertilization rates in groups with male or female partners being HBsAg-seropositive were both significantly lower than the matched controls (80.2% vs. 82.8%, P < 0.05; 76.6% vs. 84.3%, P < 0.01, respectively). HBV infection was also found to be associated negatively with fertilization rate by logistic regression analysis (odds ratios: 0.410, 95% confidence interval: 0.186–0.906, P < 0.05). However, there was no significant difference in clinical pregnancy rates between HBsAg-seropositive and HBsAg-seronegative group. These results suggest that chronic HBV infection is likely to represent a significant cause of infertility.