The biosynthetic pathway of mammalian protein asparagine (N)-glycosylation is known to produce three classes of N-glycans of the high mannose, hybrid and complex type that are abundantly expressed on glycoproteins. A fourth class of N-glycosylation known as paucimannosylation, defined as the addition of a full or partial chitobiose core to asparagine residues, is considered to be a glycosylation feature of invertebrates. However, recent literature has reported the presence of paucimannosidic proteins in humans and in mice during inflammation-related conditions, suggesting a potential immunological link to these novel glycoproteins in mammals. Given the importance of neutrophils in innate immunity, we here perform an in vitro based investigation of the biosynthesis and potential immunological functions of protein paucimannosylation in human neutrophil-like HL60 cells. Moderate to strong co-localisation of paucimannosidic epitopes, β-hexosaminidase A (Hex A) and azurophilic granule resident myeloperoxidase using immunofluorescence indicates involvement of Hex A in the synthesis of protein paucimannosylation, suggesting an azurophilic-specific localisation of paucimannosidic proteins in neutrophils. Additionally, HL60 and P.aeruginosa co-culture and the use of mass spectrometry showed that the azurophilic compartments of the neutrophil can be mobilised upon virulent stimuli, resulting in a secretion of paucimannosidic proteins into the extracellular milieu. Finally, we show that human proteins carrying these novel glycoepitopes display significant bacteriostatic activities towards specific P. aeruginosa strains under biologically relevant concentrations, as shown by the effect of purified paucimannosidic proteins on bacteria growth. Taken together, we here provide evidence supporting functionally important, previously un-described, roles of protein paucimannosylation in human neutrophils.