DNA cytosine methylation is a critical epigenetic modification that has important roles in development, cellular differentiation, genome regulation, and disease. We have investigated the role of the de novo DNA methyltransferase DNMT3B in pluripotency by constructing WA09 (H9) human embryonic stem cell (hESC) lines, where the expression of DNMT3B has been knocked-down (KD) using a lentiviral vector carrying an shRNA against DNMT3B. We have compared these DNMT3B KD hESCs to wild-type H9 hESCs, control hESCs transduced with an empty lentiviral vector, and fully differentiated human dermal fibroblasts (HDFs). We found significant differences in both the composition and patterns of cytosine methylation, most notably in the non-CpG context of CpA methylation. We observed that CpA methylation in the DNMT3B KD cells was more similar to the differentiated HDFs than the control hESCs. We also identified regions of differential CpG methylation between the DNMT3B KD and control hESCs, with the most dramatic differences in regions located in the first exons of coding regions and near CpG island shores. Moreover, we observed a significant increase in cell proliferation in the DNMT3B KD hESCs compared to control hESCs, which correlates with an increase in the expression of cell cycle-related genes. Our results suggest that DNMT3B may play a role in CpA methylation and cell cycle regulation.