Cutaneous squamous cell carcinoma (cSCC), a type of non-melanoma skin cancer (NMSC), is the most common malignancy worldwide. Thioredoxin (TXN) domain-containing protein 9 (TXNDC9) is a member of the TXN family that is important in cell differentiation. However, the biological function of this protein in cancer, particularly cSCC, is still unknown. In the present study, our experiments revealed the protective effects of TXNDC9 on UV-B-irritated cSCC cells. The initial findings showed that TXNDC9 is significantly upregulated in cSCC tissue and cells compared to normal skin tissue and keratinocytes. UV-B radiation robustly induces the expression of TXNDC9, and UV-B-induced cSCC cell death is boosted by TXNDC9 deficiency. Moreover, cSCC cells lacking TXNDC9 displayed attenuated activation of the NF-κB pathway. Additional studies by inhibiting TXNDC9 confirmed this finding, as TXNDC9 deficiency attenuated UV-B radiation-induced translocation of NF-κB p65 from the cytoplasm to the nucleus of cSCC. In conclusion, our work demonstrates the biological roles of TXNDC9 in cSCC progression and may provide a novel therapeutic target to treat cSCC in the future.