Objective To study the hepatitis B virus (HBV) vertical transmission via infected spermatozoa. Methods Eighteen male patients with HBV infection who underwent in vitro fertilization (IVF) were studied, 5 HBV negative patients were selected as the control. Fluorescence in situ hybridization (FISH) analysis using the partial-length HBV DNA as the hybridization probe was performed to explore the existence of HBV DNA in the sperm and in the host embryonic genome. Results FISH showed that 5 of 18 patients’ sperm presented positive signals and 2 of 18 embryos presented positive signals, while no positive signals were found in control group. Conclusion The HBV DNA was found in human sperm and embryos of HBV patients. These results provide direct evidence that HBV DNA could transmit to foetus via human infected spermatozoa. Objective To study the hepatitis B virus (HBV) vertical transmission via infected spermatozoa. Methods Eighteen male patients with HBV infection who underwent in vitro fertilization (IVF) were studied, 5 HBV negative patients were selected as the control. Fluorescence in situ hybridization ) analysis using the partial-length HBV DNA as the hybridization probe was performed to explore the existence of HBV DNA in the sperm and in the host embryonic genome. Results FISH showed that 5 of 18 patients’ sperm presented positive signals and 2 of 18 embryos The positive signals, while no positive signals were found in control group. Conclusion The HBV DNA was found in human sperm and embryos of HBV patients. These results provide direct evidence that HBV DNA could transmit to foetus via human infected spermatozoa.