Most street rabies virus G proteins have two N-glycosylation sites, i.e. Asn(37) and Asn(319), whereas additional sites are found in fixed (laboratory adapted) viruses. In this study, we performed a pseudotyped virus assay using G-deficient rabies virus and demonstrated that single-N-glycan additions to the G protein of street rabies virus strain 1088, which are found in adapted strains, enhanced virus production in neural and non-neural cell lines, while additions to Asn(194) or Asn(247) enhanced production greatly. Moreover, we found that N-glycan additions at Asn(194) or Asn(247) facilitated the production of cell-associated virus. In contrast, deletion of the sequon at Asn(37) reduced viral production, while a deletion at Asn(319) resulted in extensive loss of production. Furthermore, G proteins lacking an N-glycan at Asn(319) failed to fold into their correct structure and lost their fusion activity, indicating that Asn(319) N-glycosylation is important for the functional expression of street virus G proteins.