Chronic hepatitis B (CHB) remains a global health problem, carrying a high risk for progression into cirrhosis and liver failure. Molecular chaperones are involved in diverse pathophysiological processes including viral infection. However, the role of molecular chaperones in hepatitis B virus (HBV) infection and its underlying mechanisms remain unclear. Here, we identified GRP78 as one of the molecular chaperones most strongly induced by HBV in human hepatocytes. Gain- and loss-of-function analyses demonstrated that GRP78 exerted an inhibitory effect on HBV transcription and replication. Further study showed that GRP78 was involved in the activation of AKT/mTOR signaling in hepatocytes, which contributed to GRP78-mediated inhibition of HBV. Of note, HBV-upregulated GRP78 was found to play a crucial role in maintaining the survival of hepatocytes via facilitating a mild endoplasmic reticulum (ER) stress. Together, our findings suggest that HBV may sacrifice part of its replication for establishing a persistent infection through induction of GRP78, a master ER stress regulator. Targeting GRP78 may help develop to design novel therapeutic strategies against chronic HBV infection and the associated hepatocellular carcinoma.