We have previously reported that the transformation of the budding yeast with plasmids encoding the human DNA methyltransferases DNMT1 and DNMT3B cDNAs induces the mRNA of flocculin gene FLO1 and the flocculation phenotype. In the present study, we evaluated the effect of DNMT inhibitor in the transformed yeasts using a FLO1 promoter-based green fluorescent protein (GFP) reporter gene assay. The DNMT inhibitor, 5-aza-2′-deoxycytidine (5AZ), decreased GFP fluorescence driven by FLO1 promoter in DNMT-genes transformed yeast (DNMT yeast). Surprisingly, the GFP activity driven by cytosine-phosphate-guanine (CpG) motif-reduced FLO1 promoter decreased both in DNMTs gene-transformed and control strains. Yeast cells transformed with expression vector encoding a maintenance enzyme DNMT1 cDNA showed a flocculation phenotype that was associated with an enhanced mRNA level of FLO1. Bisulfite sequencing revealed methylated CpG sites at the FLO1 promoter in a control strain not expressing any DNMT transgenes, and no detectable methylation at the sites was observed in cells treated with 5AZ. These results suggest that the FLO1 promoter is endogenously de novo methylated leading to the activation of FLO1 gene transcription. Furthermore, the methylation level at the FLO1 promoter is responsible for the significant differences in FLO1 promoter-driven expression of GFP in DNMT yeast.