Nuclear factor-kappa B (NF-κB) is one of the transcription factors that play a central role in the immune response and is involved in many physiological phenomena such as acute and chronic inflammatory reactions, cell proliferation and apoptosis. It has been reported that microRNA (miRNA) binds mainly to 3'UTR of the target mRNA, suppresses its gene expression, and plays an important role in various diseases such as cancer. Therefore, the purpose of this study was to identify miRNA that regulates the activation of NF-κB signaling. We identified miR-582-5p as a significantly downregulated miRNA in inflamed mouse adipose tissue. We found miR-582-5p was suppressed in LPS-stimulated RAW264.7 cells, a macrophage-like cell line. Subsequently, S-phase kinase-associated protein 1 (SKP1), a component of the E3 ubiquitin ligase that regulates the NF-κB pathway, was detected as a potential target for miR-582-5p predicted by Targetscan. The dual luciferase reporter assay confirmed that miR-582-5p binds to the 3'UTR site of Skp1, and the transfection of miR-582-5p mimic suppresses SKP1 expression in RAW264.7 cells. In addition, miR-582-5p suppressed the production of the inflammatory cytokine TNF-α by inhibiting the degradation of IκBα, which subsequently suppressed the phosphorylation of p65 resulting in its nuclear translocation. Our findings demonstrated that exogenously applied miR-582-5p can attenuate the activation of NF-κB signaling pathway by targeting Skp1 gene expression, which provides a prospective therapeutic strategy for treating inflammatory diseases.