Posttranslational modification by the small ubiquitin-related modifier (SUMO) is one of the crucial cellular processes in Giardia lamblia, a protozoan pathogen. In this study, 5 candidate SUMO substrate proteins of G. lamblia trophozoites were chosen based on their enrichment through affinity chromatography using a SUMO-interaction motif: never in mitosis A-related kinase (NEK), aminoacyl-histidine dipeptidase (AHD), protein disulfide isomerase 2 (PDI2), alcohol dehydrogenase 3, and ornithine carbamoyltransferase. Transgenic Giardia trophozoites expressing 1 of the 5 candidate SUMO substrate proteins were constructed, and their expression was confirmed by western blot using hemagglutinin-tag. Arginine deiminase (ADI) protein was expressed in Giardia trophozoites as a positive control. Cell extracts were processed for affinity chromatography using SUMO-interaction motif resin. As expected, the SUMOylated form of ADI was detected in the affinity chromatography extracts of ADI-expressing cells. Among the 5 candidate proteins, SUMOylated forms of NEK, AHD, and PDI2 were identified in the affinity chromatography extracts. These results suggest that NEK, AHD, and PDI2 activity is modulated via SUMOylation in Giardia trophozoites.