Objective：This study aims to investigate the effects of protein kinase C-α（PKC-α）over-expression on the phosphorylation level of nuclear factor-κB-p65（NF-κB-p65）in human embryonic kidney 293T（HEK-293T）cells，and examine the inhibitory effect of sinomenine（SIN）on PKC-α/NF-κB-p65 signaling pathway. Methods：To construct the rat wild type（WT）PKC-α over-expression plasmid（pIRES2-PKC-α WT，PKC-αWT），rat PKC-α complete sequence coding（CDS）was amplified by PCR and cloned into pIRES2-EGFP. Then，alanine（A） at the site of 25 and lysine（K） at the site of 368 were mutated to glutamic（E） and arginine（R），respectively based on PKC-αWT to construct PKC-α constitutively active（CA） mutant（pIRES2-PKC-α A25E，PKC-αCA）and PKC-α dominant negative（DN） mutant（pIRES2-PKC-αK368R，PKC-αDN）. The rat wild-type NF-κB-p65 over-expression plasmid，namely pIRES2-NF-κB-p65（p65WT），was constructed by our research group previously. The plasmid of p65WT was transfected into HEK-293T cells together with the above-mentioned different PKC-α over-expression plasmids respectively. The expression and phosphorylation levels of PKC-α and NF-κB-p65 were detected by Western blot. Next，HEK-293T cells were co-transfected with the above-mentioned plasmids in different groups，and after 46 h cells were continuously treated with SIN at the dose of 50 ng/mL for 2 h. Then，the effects of SIN on phosphorylation of PKC-α and NF-κB-p65 were determined by Western blot. Results：PCR analysis and nucleotide sequencing verified that the above-mentioned PKC-α over-expression plasmids were constructed successfully. The phosphorylation of NF-κB-p65 in HEK-293T cells was increased in response to PKC-αWT and PKC-αCA over-expression，especially PKC-αCA over-expression. However，there was no significant change of NF-κB-p65 phosphorylation after PKC-αDN over-expression. SIN treatment could inhibit the phosphorylation of PKC-α in HEK-293T cells transfected with PKC-αWT，PKC-αCA and PKC-αDN. SIN could also inhibit the phosphorylation of NF-κB-p65 in HEK-293T cells caused by PKC-αWT over-expression，but not by PKC-αCA or PKC-αDN over-expression. Conclusion：Over-expression of PKC-α can promote the phosphorylation of NF-κB-p65 in HEK-293T cells. SIN treatment can down-regulate NF-κB-p65 phosphorylation through inhibition of PKC-α phosphorylation in HEK-293T cells.