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We showed here a post-translational N-glycosylation affected by the HRD1 E3 ubiquitin ligase. Both WT- and E3-defective C329S-HRD1 decreased the level of high mannose form of ABCG8, a protein that heterodimerizes with ABCG5 to control sterol balance. Meanwhile, HRD1 increased the non-glycosylated ABCG8 regardless of its E3 activity, thereby suppressing full maturation of ABCG5\/8 transporter. Pulse chase and mutational analysis indicated that HRD1 inhibits STT3B-dependent post-translational N-glycosylation of ABCG8. Whereas, HRD1 had only slight effect on the N-glycosylation status of ABCG5; rather it accelerated ABCG5 degradation in an E3 activity-dependent manner. Finally, RMA1, another E3 ubiquitin ligase, accelerated the degradation of both ABCG5 and ABCG8 <jats:italic>via<\/jats:italic> E3 activity-dependent manner. HRD1 and RMA1 may therefore be negative regulators of disease-associated transporter ABCG5\/ABCG8. 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