Hepatitis B virus (HBV)-induced hepatocellular carcinoma (HCC) is one of the most frequently occurring cancers. Hepadnaviral DNA integrations are considered to be essential agents which can promote the process of the hepatocarcinogenesis. More and more researches were designed to find the relationship of the two. In this study, we investigated whether HBV DNA integration occurred at sites of DNA double-strand breaks (DSBs), one of the most detrimental DNA damage. An 18-bp I-SceI homing endonuclease recognition site was introduced into the DNA of HepG2 cell line by stable DNA transfection, then cells were incubated in patients' serum with high HBV DNA copies and at the same time, DSBs were induced by transient expression of I-SceI after transfection of an I-SceI expression vector. By using nest PCR, the viral DNA was detected at the sites of the break. It appeared that integration occurred between part of HBVxgene and the I-SceI induced breaks. The results suggested that DSBs, as the DNA damages, may serve as potential targets for hepadnaviral DNA insertion and the integrants would lead to widespread host genome changes necessarily. It provided a new site to investigate the integration.