Objective To establish a BALB/c mouse model of influenza B virus infection, and to provide a foundation for studying the pathogenesis, mechanism of transmission of influenza B virus infection and developing new vaccines against influenza B virus. Methods Using genetic synthesis and reverse genetic technology, influenza B virus was rescued in vitro. We used the whole genome sequencing approach to validate the identity between the rescued viral genome sequences and the sequences reported in Genbank. To establish the BALB/c mouse model of influenza B virus infection, BALB/c mice were infected with 10⁵ EID₅₀ dose of the rescued virus, and the weight change, survival rate, and viral replication in the lungs were analyzed. Results We successfully rescued influenza B virus B/Yamagata/16/88 in vitro, and this virus was named B-S9. The genome sequencing results showed that the genome sequences of B-S9 was consistent with the GenBank-reported sequences. BALB/c mice were artificially infected with B-S9, and no death due to infection was observed. The above results indicated that B-S9 is of low pathogenicity to the BALB/c mice. The mice infected with B-S9 showed body weight decline in 3 days post inoculation (dpi) but restored in 8 dpi. The virus titers could be detected in the lungs of mice infected with B-S9 on dpi 3 and dpi 6, respectively. Furthermore, the virus titer in the mouse lungs on dpi 3 was 132 times higher than that on dpi 6. Conclusions A reverse genetic system of influenza B virus B/Yamagata/16/88 is successfully established, and a BALB/c mouse model of influenza B virus infection is established. To date, studies of influenza B virus are limited at home and abroad. The establishment of this reverse genetic system provides not only a platform for studying the pathogenesis and mechanism of transmission of influenza B virus, but also provides a way for developing new live-attenuated influenza B virus vaccine.