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The mitochondrial calcium and downstream proline-rich tyrosine Kinase-2(PyK2)signaling pathway are critic al to hepatitis B virus(HBV)replication，and the endoplasmic reticulum(ER)plays an important role in intracellular calcium regulation. To investigate the role of ER in HBV replication，the HBV genome transfected HepG2.2.15 cells were treated by cyclosporine A(CsA)，cyclopiazonic acid(CPA)，ryanodine and U73122，which are all specific blockers of calcium channels located in either ER or mitochondria. The HBV replication level was evaluated by two methods：slot blot hybridization analysis of intracellular HBV DNA and real-time polymerase chain reaction(PCR)analysis of secreted HBV DNA in supernatant; the activation of PyK2 kinase was detected by western blot analysis. Results indicated that the HBV replication was inhibited when mitochondrial permeability transition pore，ER Ca2+-ATPase and ER inositol 1，4，5-trisphosphate receptor(IP3R)were blocked by CsA，CPA and U73122，respectively; but not inhibited when ER ryanodine receptor was blocked by ryanodine. The PyK2 phosphorylation level declined after treatment of 2μg/ml CsA，5μM CPA and 25μM U73122，but not changed apparently after 50μM ryanodine treatment.