The Ebola virus (EBOV) trimeric envelope glycoprotein (GP) precursors are cleaved into the receptor-binding GP and the fusion-mediating GP subunits and incorporated into virions to initiate infection. GP and GP form heterodimers that have 15 or two -glycosylation sites (NGSs), respectively. Here we investigated the mechanism of how-glycosylation contributes to GP expression, maturation, and function. As reported before, we found that, although GP NGSs are not critical, the two GP NGSs, Asn and Asn, are essential for GP function. Further analysis uncovered that Asn and Asn regulate GP processing, demannosylation, oligomerization, and conformation. Consequently, these two NGSs are required for GP incorporation into EBOV-like particles and HIV type 1 (HIV-1) pseudovirions and determine viral transduction efficiency. Using CRISPR/Cas9 technology, we knocked out the two classical endoplasmic reticulum chaperones calnexin (CNX) and/or calreticulin (CRT) and found that both CNX and CRT increase GP expression. Nevertheless, NGSs are not required for the GP interaction with CNX or CRT. Together, we conclude that, although Asn and Asn are not required for EBOV GP expression, they synergistically regulate its maturation, which determines its functionality.