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STUDY QUESTION What is the impact of male hepatitis B virus (HBV) infection on sperm quality, embryonic development, and assisted reproductive outcomes? SUMMARY ANSWER Male HBV infection did not affect assisted reproductive outcomes, but HBV is capable of impairing human sperm and embryo formation in the early stages following fertilization. WHAT IS KNOWN ALREADY HBV is found in germ cells and early embryos of patients with HBV. HBV may impair human sperm function via increasing reactive oxygen species. STUDY DESIGN, SIZE, DURATION We conducted a retrospective cohort study of 1581 infertile couples, including 496 male patients clinically confirmed to have hepatitis B infection, and a laboratory study of effects of HBV proteins on early embryos, using human embryonic stem cells (hESCs), human sperm, and golden hamster oocytes. PARTICIPANTS/MATERIALS, SETTING, METHODS In total, 1581 infertile couples (24–40 years of age) who were admitted to a reproductive medicine center to undergo ART for the first time from January 2019 to November 2021 were selected as the study subjects. The case group was composed of 469 couples with hepatitis B surface antigen (HBsAg)-seropositive men and seronegative women (368 for IVF and 101 for ICSI treatment). The negative control group was composed of 1112 couples where both men and women were seronegative for hepatitis B antigen. We divided these couples into three comparison groups (IVF/ICSI, IVF, and ICSI). IVF of human sperm and hamster oocytes was used to evaluate the influence of the HBV HBs protein on formation of 2-cell embryos. Mitochondrial membrane potential (MMP) of hESCs was assayed via a fluorescence intensity system. Immunofluorescence staining of the phosphorylated histone H2A.X was applied to identify DNA damage to hESCs caused by the HBV X (HBx) protein. MAIN RESULTS AND THE ROLE OF CHANCE Sperm concentration, total sperm number, and sperm with normal morphology were decreased in the couples with HBV-infected males in couples who were undergoing IVF/ICSI (male HBV(+) vs control: 469 vs 1112 individuals; sperm number, P  <   0.01; normal sperm morphology, P  <   0.01), IVF (368 vs 792; sperm number, P  <   0.01; normal sperm morphology, P  ≤   0.05), and ICSI (101 vs 306; sperm number, P  <   0.01; normal sperm morphology, P  <   0.001). There was no significant difference in the number of embryo cleavages, blastocyst formation, biochemical pregnancy rate, clinical pregnancy rate, and live-birth rate between case and control groups. The 2PN fertilization rate in IVF/ICSI (P  <   0.01) and ICSI (P  <   0.05) couples, and the number of 2PN-fertilized oocytes in IVF (P  <   0.001) couples were lower in couples with male HBV infection compared to control couples. HBV HBs protein reduced the MMP of human sperm and decreased 2-cell embryo formation in IVF of human sperm and zona-free-hamster oocyte. A reduction in fluorescence intensity and immunofluorescence staining of phosphorylated histone H2A.X indicated that HBx caused MMP impairment and DNA damage in human early embryonic cells, respectively. LARGE SCALE DATA N/A. LIMITATIONS, REASONS FOR CAUTION HBV can be examined in samples of sperm or discarded IVF early embryos from HBsAg-seropositive men and seronegative women. The hESC model in vitro may not fully mimic the natural embryos in vivo. WIDER IMPLICATIONS OF THE FINDINGS This study furthers our understanding of the influence of male HBV infection on embryonic development. Our results suggest that a semen-washing process may be necessary for male patients with HBV undergoing ART to minimize the potential negative effects of HBV infection on the early embryo. STUDY FUNDING/COMPETING INTEREST(S) This work was funded by grants from the National Natural Science Foundation of China, grant numbers 81870432 and 81570567 to X.Z. 81571994 to P.S. and 81950410640, the Natural Science Foundation of Guangdong Province, China (No. 2023A1515010660 to X.Z.), and the Li Ka Shing Shantou University Foundation (Grant No. L11112008). The authors have no conflicts of interest. [ABSTRACT FROM AUTHOR]