Avian leukemia virus subgroup J (ALV-J) causes various diseases associated with tumor formation, decreased fertility and immunosuppression, resulting in significant economic losses in the poultry industry globally. Virus usually exploits the host cellular machinery for their replication. Although there are increasing evidences for the cellular proteins involving viral replication, the interaction between ALV-J and host proteins leading to the pivotal steps of viral life cycle are still unclear. Here, we reported that the heat shock protein 70 (Hsp70) plays a critical role during ALV-J infection by interacting with gp37 and P32. Changing the expression of Hsp70 affects the replication of ALV-J in host cells, and inhibitory of Hsp70 using the specific inhibitors JG-98 or Pifithrin-μ significantly reduced viral replication. This study revealed the effect of host Hsp70 on ALV-J replication, providing insights for further studies of the molecular mechanism of ALV-J infection.