Endoplasmic reticulum (ER) alpha-glucosidase inhibitors block the trimming of N-linked glycosylation and thus prevent the production of several viruses. The present study investigates the antiviral effects of the alpha-glucosidase and alpha-mannosidase inhibitors (castanospermine, 1-deoxynojirimycin, bromoconduritol, deoxymannojirimycin and swainsonine) on human parainfluenza virus type 3 (HPIV3). The alpha-glucosidase inhibitors (castanospermine, 1-deoxynojirimycin) in recombinant expression systems reduced the surface and intracellular expression of both HPIV3 F and HN proteins. On the other hand, alpha-mannosidase inhibitors prevented processing of the oligosaccharides on HPIV3 glycoproteins into the complex form. Consequently, alpha-glycosidase inhibitors (castanospermine and 1-deoxynojirimycin) significantly inhibited viral fusion activity. We demonstrated that the alpha-glucosidase inhibitors (castanospermine and 1-deoxynojirimycin) reduced the infectivity of newly released viral particles. We postulate that alpha-glucosidase inhibitors can prevent the first steps of HPIV3 envelope glycoprotein processing and that the inhibition of glucose trimming has antiviral effects.