Capsaicin, a natural product of the Capsicum species of red peppers, is known to induce apoptosis and suppress growth. Non-steroidal anti-inflammatory drug-activated gene-1 (NAG-1) is a cytokine associated with pro-apoptotic and antitumorigenic property in colorectal and lung cancer. Our data demonstrate that capsaicin leads to induction of apoptosis and up-regulates NAG-1 gene expression at the transcriptional level. Overexpression of CCAAT/enhancer binding protein beta (C/EBPbeta) caused a significant increase of basal and capsaicin-induced NAG-1 promoter activity. We subsequently identified C/EBPbeta binding sites in the NAG-1 promoter responsible for capsaicin-induced NAG-1 transactivation. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay confirmed binding of C/EBPbeta to the NAG-1 promoter. Capsaicin treatment resulted in an increase of phosphorylated serine/threonine residues on C/EBPbeta, and the immunoprecipitation study showed that capsaicin enhanced binding of C/EBPbeta with glycogen synthase kinase 3beta (GSK3beta) and activating transcription factor 3 (ATF3). The phosphorylation and interaction of C/EBPbeta with GSK3beta and ATF3 are decreased by the inhibition of the GSK3beta and Protein Kinase C pathways. Knockdown of C/EBPbeta, GSK3beta or ATF3 ameliorates NAG-1 expression induced by capsaicin treatment. These data indicate that C/EBPbeta phosphorylation through GSK3beta may mediate capsaicin-induced expression of NAG-1 and apoptosis through cooperation with ATF3 in human colorectal cancer cells.