Hepatitis B virus (HBV) infection is a major public health problem by affecting 350 million people worldwide. The mechanisms that regulate HBV gene expression and viral replication remain poorly understood. HBx is known as the central regulator for HBV replication and is associated with the CUL4-DDB1 ubiquitin ligase through H-box motif. Here, we show that blocking the activity of DDB1 by RNA interfering inhibited viral production and gene expression of HBV, and direct association of HBx with DDB1 promoted viral activities, indicating that DDB1 function is required for viral production. On the other hand, HBx interfered with DDB1-dependent polyubiquitination of PRMT1, arginine methyltransferase 1, suggesting that HBx can also block the function of a subset of CUL4-DDB1 E3 ligases. Thus, we conclude that HBx regulates the function of DDB1 in both positive and negative manners in the context of distinct CUL4-DDB1 complexes and plays different roles in HBV replication cycle.