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Objective: This study aimed to clarify the infection state of early embryos by HBV and the effect of HBV infection on embryo development. Methods: In vitro culture was carried out on the fresh surplus embryos from infertile couples on the third day (D3) who received routine in vitro fertilization and embryo transfer (IVF-ET) treatment. The embryo cells were infected in vitro by the serum of HBV infected patients with different viral loads. HBsAg quantity in the supernatant secreted by embryo and HBV mRNA in embryo cells were detected. The development of embryo cells after HBV infection was evaluated by embryo morphological parameter score and fragment score in embryo cells. Results: From May 2013 to July 2015, 238 cases of D3 fresh surplus embryos from 120 infertile couples and 128 samples of embryo culture supernatant were collected. The HBsAg positive rate in the supernatant of embryos in the high viral load infection group was significantly higher than that in the low viral load infection group (40.0% vs.15.6%, p<0.05). HBV mRNA was detected in 2 of 17 HBsAg positive embryos. The scores of embryos in the high load HBV culture solution group were higher than those in the low load group (p=0.04). There was one formed blastocyst in low load HBV culture group, but there was no blastocyst formation in all embryo cells of high load group (p<0.001).Conclusion: The results suggest that embryo cells can be directly infected by HBV in vitro. HBV infection affects the development of embryos.