Mannosyl phosphorylceramide (MIPC) is a membrane lipid classified as a complex sphingolipid in Saccharomyces cerevisiae. MIPC is synthesized by two redundant enzymes, Sur1/Csg1 and Csh1, in the Golgi lumen. MIPC consists of five subtypes (A, B', B, C, and D-type) according to the position and number of hydroxyl groups on the ceramide moiety. Sur1 exerts higher impact on synthesis of MIPC-B and MIPC-C than Csh1. In this study, we elucidated the roles played by N-glycans attached to Sur1 and Csh1, and dissected the mechanisms underlying substrate recognition by these two enzymes. Sur1 carries an N-glycan on Asn-224, while Csh1 has N-glycans on Asn-51 and Asn-247. Although intracellular proteins usually harbor core-type N-glycans, the N-glycan on Asn-51 of Csh1 exhibited a unique mannan-like structure containing a long backbone of mannose. Sur1 N224Q and Csh1 N51Q mutants exhibited a decrease in the activity to synthesize specific MIPC subtypes for each enzyme, suggesting that these N-glycans play a role in substrate recognition through their catalytic domains. Moreover, ectopic insertion of an N-glycosylation consensus sequence (NST) at codon 51 of Sur1 (Sur1-NST51) resulted in an artificial modification with mannan, which markedly decreased protein stability. Our results suggest that the diminished stability of the Sur1-NST51 mutant protein could be attributable to potential structural alterations by the mannan. Collectively, the present study reveals essential luminal domains of Sur1 and Csh1 that dictate substrate specificity and/or the protein stabilities via mannan modification.