Invertase SUC2 is well-studied as a model hydrolase for carbohydrate catabolism in yeast strains. However, little is known about the ubiquitination of SUC2 and its contribution on the enzyme. In this paper, we demonstrated for the first time, that invertase SUC2 was ubiquitinated at three lysine residue sites using mass spectrometry-based proteomics. The role of ubiquitination was investigated through site-directed mutagenesis. The K185, K312 and K430 were changed to arginine, respectively. The expression level, activity and stability of all the SUC2 isoforms together with the wild-type one were measured and compared. Results showed that ubiquitination of SUC2 could not lead to the degradation of the protein. Interestingly, ubiquitination of SUC2 in the site of 185 contributes to the high enzyme activity, and ubiquitin-tagged residue K185 in SUC2 is probably a novel active-site residue in invertase SUC2. This study provides an insight into the role of post-translational modifications in regulating the stability and activity of enzymes.