Abnormal DNA methylation is one of the important characteristics in tumor cells. Apoptosis plays an essential role in cell survival and processing. It is not clear whether DNA methyltransferases (DNMTs) change in apoptosis and how DNMTs are regulated in apoptosis. In this study, we found that SMMC-7721 or BEL-7404 cells were induced to apoptosis by STS, meanwhile the DNMT3B protein and mRNA level were decreased. To explore the mechanism of DNMT3B down-regulation, we found that the mRNA decay was not changed and core promoter activity of DNMT3B gene was decreased in STS-induced apoptosis. In order to figure out the signal molecule involved in transcriptional regulation of DNMT3B gene by STS, p-JNK, p-ERK, and p-p38 were examined. In STS-induced apoptosis p-JNK level was increased, and p-ERK and p-p38 were decreased. Furthermore, the inhibitor of p-JNK significantly alleviated the decline of DNMT3B protein. We also found that the siRNA of DNMT3B strengthened the cleavage of PARP and pro-caspase-3 as well as up-regulated the p16 gene expression in STS-treated cells. We concluded here that STS-regulated DNMT3B gene expression via p-JNK and down-regulation of DNMT3B-mediated STS-induced apoptosis through the up-regulation p16 expression.