Viruses are considered to use vesicular trafficking in infected cells, but the details of assembly/release pathways of hepatitis B virus (HBV) are still unknown. To identify key regulators of HBV production, we performed short interfering RNA (siRNA) screening for Rab proteins, which are considered to act as molecular switches in vesicular trafficking using HepG2.2.15 cells. Among 62 Rab proteins, the suppression of Rab5B most significantly increased HBV DNA in the culture supernatant. Surprisingly, 5 days after the transfection of Rab5B siRNA, HBV DNA in the supernatant was increased more than 30-fold, reflecting the increase of infectious HBV particles. Northern blotting showed that transcription of 2.4/2.1-kb mRNA coding envelope proteins containing large hepatitis B surface protein (LHBs) was increased. Analysis of hepatocyte nuclear factors (HNFs) showed that transcription of HNF4α, which is known to enhance 2.4-kb mRNA transcription, was regulated by Rab5B. Also, it was revealed that LHBs had accumulated in the endoplasmic reticulum (ER) after Rab5B depletion but not in the multivesicular body (MVB), which is thought to be an organelle utilized for HBV envelope formation. Therefore, it was considered that Rab5B is required for the transport of LHBs from the ER to MVB. Immunofluorescent microscopy showed that HBs proteins, including LHBs, colocalized with HBc in the ER of Rab5B-depleted cells, suggesting that HBV envelopment occurs not only in the MVB but also in the ER. In conclusion, Rab5B is a key regulator of HBV production and could be a target of antiviral therapy.IMPORTANCE HBV infection is a worldwide health problem, but the mechanisms of how HBV utilizes cellular machinery for its life cycle are poorly understood. In particular, it has been unclear how the viral components and virions are transported among the organelles. The HBV budding site has been reported to be the ER or MVB, but it has not been clearly determined. In this study, siRNA-based screening of Rab proteins using HBV-expressing cells showed that Rab5B, one of the Rab5 isoforms, has important roles in late steps of the HBV life cycle. Although Rab5 is known to work on early endosomes, this study showed that Rab5B plays a role in the transport of LHBs between the ER and MVB. Furthermore, it affects the transcription of LHBs. This is the first report on the mechanisms of HBV envelope protein transport among the organelles, and the results provide important insights into the therapeutic control of HBV infection.