We aimed to explore the effects of Inflammatory cytokines (IL-1β, IFN-γ, TNF-α) on pancreatic β-cells. CCK-8 assay showed that the cell viability decreased after 24 hr treatment of TNF-α, 48 hr of IFN-γ, and 84 hr of IL-1β. EdU assay illustrated that after 24 hr treatment, there were significantly reduced EdU-labeled red fluorescence cells in TNF-α group while not in IFN-γ and IL-1β groups. Flow Cytometry results displayed that TNF-α and IFN-γ groups increased apoptosis while IL-1β group did not. Cell apoptosis results found that there was an increase in the S-phase population of IL-1β and TNF-α groups, however, there was no significant difference in cell cycle between IFN-γ group and the control. TEM images showed that there were reduction in the number of granules and mitochondria in IL-1β and IFN-γ groups, in particular paucity of insulin granules and mitochondria in TNF-α group. Radioimmunoassay results presented that TNF-α inhibited glucose-induced insulin secretion, while there were no significant changes in IL-1β and IFN-γ groups when compared with the control. Metabolomic analysis found amino acid metabolism and Krebs cycle were the most robust altered metabolism pathways after inflammatory cytokines treatments. Overall, the altered amino acid metabolism and Krebs cycle metabolism might be important mechanisms of TNF-α induced mouse pancreatic β-cells dysfuction.