Trimming of the N-glycans attached to the envelope proteins of hepatitis B virus (HBV) is required in different steps of the viral life cycle. Inhibition of the host enzymes alpha-glucosidases, involved in the endoplasmic reticulum (ER)-associated processing of the N-linked glycans, results in misfolding of the HBV envelope proteins, prevention of HBV secretion and accumulation of viral DNA within infected cells. However, the impact of these effects on HBV morphogenesis and infectivity of the viral particles that are still released from cells with inhibited alpha-glucosidase has not been addressed so far. Using N-butyldeoxynojirimycin (NB-DNJ), a competitive inhibitor of the ER alpha-glucosidases, we analyzed the role of these enzymes on HBV assembly and infectivity of the virions released from HepG126.96.36.199 cells. HBV secreted from drug-treated cells contained an envelope with altered composition of the disulfide-linked oligomers and no detectable middle (M) protein. These molecular changes had a significant effect on HBV infectivity, reducing it to 20% compared to controls, for the highest concentrations of NB-DNJ used. Our data show for the first time that an active alpha-glucosidase activity is crucial for production of infectious HBV and provide new insights into the controversial role of the M protein in this process.