Tripartite motif-containing protein 21 (Trim21) is an E3 ubiquitin-protein ligase that plays pivotal roles in various diseases. However, its role in mediating keratinocyte inflammation, which is a hallmark of psoriasis, has not been thoroughly elucidated.To clarify whether Trim21 plays a pivotal role in regulating keratinocyte inflammation in psoriasis, while focusing on identifying key Trim21 substrates involved in mediating proinflammatory cytokine and chemokine production.Cytokine and chemokine secretion was examined by quantitative real-time PCR (qPCR) in Trim21-knockdown human keratinocytes. Downstream pathways and substrates of Trim21 were evaluated using immunoblotting, immunoprecipitation and immunofluorescence. The influence of Trim21 ubiquitination on its substrates was tested by in vitro ubiquitination assay, immunoprecipitation and immunofluorescence. The effectiveness of targeting Trim21 for psoriasis treatment was assessed in vivo with haematoxylin and eosin (H&E) staining, immunofluorescence and qPCR.Knocking down Trim21 expression alleviated keratinocyte inflammation. Trim21 colocalized with p65/Nuclear factor-κB (NF-κB) in the cytosol and physically bound and ubiquitinated p65 via a lysine 63 (K63) linkage. Instead of changing p65 protein stability, Trim21 enhanced the interaction of p65 with IκB kinase (IKK), which promoted p65 phosphorylation, nuclear transport and downstream gene activation. Finally, both in vitro and in vivo experiments verified that topical application of Trim21-specific small interfering RNA (siRNA) markedly ameliorated imiquimod (IMQ)-induced psoriasis-like lesions.Our study confirms that upregulated Trim21 in psoriatic epidermis ubiquitylates p65 and activates the NF-κB pathway, which promotes keratinocyte inflammation. Hence, Trim21 represents a potential target for psoriasis treatment.