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Nuclear factor-κB (NF-κB) is an important transcription factor. While the NF-κB signaling pathway is modulated by many microRNAs (miRNAs), very few have been reported to target NF-κB1 gene directly. In this study, we used multiple miRNA target prediction programs to predict miRNAs with putative NF-κB1 3′-untranslated region (UTR) binding sites. miR-183 was strongly implicated and experimentally validated by reporter assays. The results showed a reduced expression of the NF-κB1 3′UTR containing luciferase vector by ∼30%, which was comparable to the reduction by miR-9 (the only known miRNA targeting the NF-κB1 3′UTR). Mutagenesis of the miR-183 seed region binding sequence in the NF-κB1 3′UTR abolished the inhibitory effect of miR-183, as noted by the NF-κB1 3′UTR-containing reporter. Moreover, similar to miR-9, miR-183 could down-regulate the expression of the reporter …