E1, along with E(rns) and E2 is one of the three envelope glycoproteins of Classical Swine Fever Virus (CSFV). Previously we showed that glycosylation status of virulent CSFV strain Brescia E2 or E(rns) affects virus virulence. Here, the three putative glycosylation sites of E1 were serially removed by means of site directed mutagenesis of a CSFV Brescia infectious clone (BICv) and their effect on virulence assessed in swine. Removal of all three putative glycosylation sites in E1, at CSFV positions N500, N513 and N594, yielded nonviable progeny, while single or dual site mutants excluding N594 were viable. Individual N594A (E1.N3 virus) or combined N500A/N513A (E1.N1N2 virus) substitutions resulted in BICv attenuation. Furthermore infection with E1.N3 or E1.N1N2 viruses efficiently protected swine from challenge with virulent BICv at 3 and 28 days post-infection. As previously observed with E(rns) and E2 and here with E1 data suggest that modification of glycosylation patterns could be used for developing CSFV live-attenuated vaccines.