Ceruloplasmin (CP) is mainly synthesized by hepatocytes and plays an essential role in iron metabolism. Previous reports have shown that CP levels correlate negatively with disease progression in patients with chronic hepatitis B. However, the function of CP in the hepatitis B virus (HBV) life cycle and the mechanism underlying the above correlation remain unclear. Here, we report that CP can selectively inhibit the production of extracellular HBV virions without altering intracellular viral replication. HBV expression can also downregulate the expression of CP. Knockdown of CP using small interfering RNA significantly increased the level of extracellular HBV virions in both Huh7 and HepG2.2.15 cells, while overexpression of CP decreased this level. Mechanistically, CP could specifically interact with the HBV middle surface protein (MHB). Using an HBV replication-competent clone unable to express MHBs, we demonstrated that the overexpression of CP did not affect the production of extracellular HBV virions in the absence of MHBs. Furthermore, introduction of an MHB expression construct could rescue the impairment in virion production caused by CP. Taken together, our results suggest that CP may be an important host factor that targets MHBs during the envelopment and/or release of virions.