DNA methylation, which affects gene expression and chromatin stability, is catalyzed by DNA methyltransferases (DNMTs) of which DNMT1 possesses most abundant activity. PI3K/PKB pathway is an important pathway involved in cell proliferation, viability, and metabolism and often disrupted in cancer. Here we investigated the impact of PKB on DNMT1 and DNA methylation. Positive correlation between PKB-Ser473-phosphorylation and DNMT1 protein level in 17 human cell lines (p<0.01) and in 27 human bladder cancer tissues (p<0.05) was found. With activator, inhibitor, siRNA and constitutively active or dominant-negative plasmids of PKB, we found that PKB increased the protein level of DNMT1 without coordinate mRNA change, which was specific rather than due to cell-cycle change. PKB enhanced DNMT1 protein stability independent of de novo synthesis of any protein, which was attributed to down-regulation of N-terminal-120-amino-acids-dependent DNMT1 degradation via ubiquitin–proteasome pathway. Gsk3β inhibitor rescued the decrease of DNMT1 by PKB inhibition, suggesting that Gsk3β mediated the stabilization of DNMT1 by PKB. Then role of PKB regulating DNMT1 was investigated. Inhibition of PKB caused observable DNA hypomethylation and chromatin decondensation and DNMT1 overexpression partially reversed cell growth inhibition by PKB inhibition. In conclusion, our results suggested that PKB enhanced DNMT1 stability and maintained DNA methylation and chromatin structure, which might contribute to cancer cell growth.