Objective: In this study, we used reverse genetics method to rescue a recombinant H1N1 pdm09 viruses. Methods: The six internal gene segments of A/Puerto Rico/8/1934 (PR8) virus were amplified by RT-PCR, HA and NA of A/Wisconsin/588/2019 (H1N1) were synthesized. The above eight gene segments were cloned into pHW2000 bidirectional expression vector, respectively. The plasmids were co-transfected into 293T cells to rescue the recombinant virus. The recombinant virus was identified by RT-PCR and hemagglutination test. The viral growth curve in MDCK cells was drawn. The viral pathogenicity was evaluated in mice. Results: The recombinant A/Wisconsin/588/2019 virus was rescued. The viral titers of the recombinant virus on MDCK cells were 105.38TCID50/mL. The hemagglutination titers reached 29 on eggs. The median lethal dose to mice was LD50=10-1.38/50μL. Conclusion: A recombinant H1N1 pdm09 virus was successfully rescued. This study provides a new idea for the rapid rescue of influenza viruses by reverse genetics method.