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It has long been thought that iminosugar antiviral activity is a function of inhibition of endoplasmic reticulum-resident α-glucosidases, and on this basis, many iminosugars have been investigated as therapeutic agents for treatment of infection by a diverse spectrum of viruses, including dengue virus (DENV). However, iminosugars are glycomimetics possessing a nitrogen atom in place of the endocyclic oxygen atom, and the ubiquity of glycans in host metabolism suggests that multiple pathways can be targeted via iminosugar treatment. Successful treatment of patients with glycolipid processing defects using iminosugars highlights the clinical exploitation of iminosugar inhibition of enzymes other than ER α-glucosidases. Evidence correlating antiviral activity with successful inhibition of ER glucosidases together with the exclusion of alternative mechanisms of action of iminosugars in the context of DENV infection is limited. Celgosivir, a bicyclic iminosugar evaluated in phase Ib clinical trials as a therapeutic for the treatment of DENV infection, was confirmed to be antiviral in a lethal mouse model of antibody-enhanced DENV infection. In this study we provide the first evidence of the antiviral activity of celgosivir in primary human macrophages in vitro, in which it inhibits DENV secretion with an EC50 of 5 μM. We further demonstrate that monocyclic glucose-mimicking iminosugars inhibit isolated glycoprotein and glycolipid processing enzymes and that this inhibition also occurs in primary cells treated with these drugs. By comparison to bicyclic glucose-mimicking iminosugars which inhibit glycoprotein processing but do not inhibit glycolipid processing and galactose-mimicking iminosugars which do not inhibit glycoprotein processing but do inhibit glycolipid processing, we demonstrate that inhibition of endoplasmic reticulum-resident α-glucosidases, not glycolipid processing, is responsible for iminosugar antiviral activity against DENV. Our data suggest that inhibition of ER α-glucosidases prevents release of virus and is the primary antiviral mechanism of action of iminosugars against DENV., Author Summary Current treatment of dengue virus infection is supportive; however, iminosugars have been widely investigated as an antiviral strategy. The means by which these molecules are thought to exert their antiviral effects is through inhibition of host-resident glycoprotein processing enzymes, the endoplasmic reticulum-resident α-glucosidases, but many iminosugars are also capable of inhibiting host glycolipid processing and are utilized clinically for the treatment of lysosomal storage disorders such as Gaucher’s and Niemann-Pick type C diseases. The work presented here is the first to conclusively differentiate the antiviral properties of these two major mechanisms of action of iminosugars, and our data support the long-standing hypothesis that inhibition of glycoprotein processing is the essential antiviral property of iminosugars in the case of dengue virus infection. These results indicate that further development of iminosugars as dengue antivirals should focus on optimization of glycoprotein inhibition efficacy with reduction or elimination of glycolipid modulating properties to minimize off-target effects. These results are supported by the in vitro and in vivo efficacy of the bicyclic iminosugar, celgosivir, which we demonstrate to lack capacity for inhibition of glycosphingolipid processing.