Natural honey contains glycoconjugates as minor components. We characterized acacia honey glycoconjugates with molecular masses in the range of 2–5 kDa. The glycoconjugates were separated by RP-HPLC into three peaks (termed RP-2-5 k-I, RP-2-5 k-II, and RP-2-5 k-III) which demonstrated paralyzing effects on the model nematode C. elegans (ED50 of 50 ng glycoconjugates/μL). To examine molecular mechanisms underlying the nematicidal effects of honey glycoconjugates, expressional analyses of genes that are essential for the growth, development, reproduction, and movement of C. elegans were carried out. Quantitative PCR-based assays showed that these molecules moderately regulate the expression of genes involved in the citric acid cycle (mdh-1 and idhg-1) and cytoskeleton (act-1 and act-2). MALDI-ToF-MS/MS analysis of RP-HPLC peaks revealed the presence of paucimannose-like N-glycans which are known to play important roles in invertebrates e.g., worms and flies. These findings provided novel information regarding the structure and nematicidal function of honey glycoconjugates. Display omitted •Glycoconjugates of honey showed C. elegans paralyzing activity.•Exposure of honey glycoconjugates caused altered regulation of C. elegans genes transcripts involved in the citric acid cycle, cytoskeleton, body morphogenesis, embryo development.•Characterization of nematicidal glycoconjugates from honey by mass spectrometry revealed paucimannose like N-Glycans which are known to play important roles in survival of invertebrates e.g., worms and flies.