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Proinflammatory cytokines and NO play crucial roles in islet β-cells dysfunction. Though anti-inflammatory effects of kaempferol were revealed in several studies, the detailed mechanisms remain unclear. This study explored protective actions of kaempferol in interleukin-1β-treated RINm5F β-cells. Kaempferol significantly inhibited NO generation, iNOS protein, and iNOS mRNA level. Promoter study, EMSA, and κB-dependent reporter assay showed that kaempferol inhibited NF-κB-mediated iNOS gene transcription. Also, we found that kaempferol accelerated iNOS mRNA instability in iNOS 3′-UTR construct and actinomycin D chase studies. Additionally, kaempferol reduced iNOS protein stability in cycloheximide chase study and it inhibited NOS enzyme activity. Kaempferol inhibited ROS generation and preserved cell viability, and it improved insulin release. These findings suggest that kaempferol appears to be helpful in protecting islet β-cells, thereby supports kaempferol as a supplementary therapeutic candidate in inhibiting the incidence and progression of diabetes mellitus.