The SR-domain protein kinase (SRPK) 1 and 2 are two important kinases involved in cellular RNA splicing. Recently, it was suggested that these two kinases, which could bind to the hepatitis B virus (HBV) core protein, might be the major cellular kinases that phosphorylate the core protein to regulate HBV replication. In this report, we tested the role of SRPK1 and SRPK2 in HBV replication and found that both of them could suppress HBV replication by reducing the packaging efficiency of the pgRNA without affecting the formation of the viral core particles. This suppressive effect of SRPK1 and SRPK2 on HBV replication cannot be explained by their phosphorylation activities on the HBV core protein as the over-expression of these two kinases had no detectable effects on HBV core protein phosphorylation in vivo and their mutants that lacked the kinase activity could still suppress HBV DNA replication. Thus, these findings demonstrate a negative role of SRPK1 and SRPK2 in the regulation of HBV replication through a mechanism not involving the phosphorylation of the core protein.