Vesicular trafficking plays a critical role in the survival of the human gut pathogenGiardia lambliaas it drives nutrient uptake and morphological stage transition. Unlike most eukaryotes,Giardiahas a minimal vesicular trafficking machinery. Herein, we report a rare exception to this minimalism wherein two paralogues of NSF, a crucial factor driving vesicular trafficking by uncoupling the cis-SNARE bundle, are present in this unicellular parasite. While GlNSF114776and GlNSF112681share very high sequence homology, they are likely to have distinct cellular roles as they exhibit differences in their affinities towards the Glα-SNAPs and display non-overlapping distribution in encysting trophozoites. Under multiple stress conditions (nutritional, oxidative and nitrosative), while GlNSF112681remains at peripheral vesicles, GlNSF114776relocalizes to the anterior flagella-associated striated fibres, indicating a possible role in regulating flagellar motility. At this location, GlNSF114776is likely to perform a 20S complex independent function as neither Glα-SNAPs nor GlSNAREs are present there. The two paralogues are likely needed for stress adaptation as both copies have also been retained inGiardiagenomes isolated from clinical samples. This non-canonical function of the GlNSF114776may have evolved to support the unique architecture and motility of the anterior flagella.