Aberrant cytosine methylation is known to be associated with cancer development. Here, we assessed how common cancer chemotherapeutic agents perturb cytosine methylation in Jurkat-T acute lymphoblastic leukemia cells. We tested six antitumor agents and found that cyclophosphamide induced the most pronounced increase in global DNA cytosine methylation after a 24-h treatment. Long-term treatment with cyclophosphamide led to a time-dependent increase in cytosine methylation level with up to 4 days of treatment, and the extent of cytosine methylation returned to normal level after 8 days. The trend of change in DNA methylation level paralleled that of the expression level of DNMT1 protein, whereas no significant increase in DNMT1 mRNA level was observed. Previous studies showed that the stability of endogenous DNMT1 protein is regulated by lysine methylation through histone lysine methyltransferase Set7 and lysine-specific demethylase 1 (LSD1), with the methylated DNMT1 being the target for proteasomal degradation. We observed that the elevated expression of DNMT1 protein at 4 days of treatment was correlated with the increased expression of LSD1 protein and with the decreased frequency of K142 methylation in DNMT1. Taken together, our results showed that cyclophosphamide perturbed temporarily global cytosine methylation in Jurkat-T cells via regulation of the lysine methylation level in DNMT1.