Disruption of the DNA methylation landscape is one of the most common features of human tumors. However, genetic alterations of DNA methyltransferases (DNMTs) have not been described in carcinogenesis. Herein, we show that pancreatic and breast cancer cells undergo gene amplification of the DNA methyltransferase 3B (DNMT3B). The presence of extra copies of the DNMT3B gene is linked to higher levels of the corresponding mRNA and protein. Most importantly, the elevated gene dosage of DNMT3B is associated with increased resistance to the growth‐inhibitory effect mediated by DNA demethylating agents. In particular, cancer cells harboring DNMT3B gene amplification are less sensitive to the decrease in cell viability caused by 5‐azacytidine (Vidaza), 5‐aza‐2‐deoxycytidine (Decitabine), and SGI‐1027. Overall, the data confirm DNMT3B as a bona fide oncogene in human cancer and support the incorporation of the DNMT3B copy number assay into current clinical trials assessing the efficacy of DNA demethylating drugs in solid tumors. © 2011 Wiley‐Liss, Inc.