Monoamine oxidase (MAO) A and B catalyze the oxidative deamination of neuroactive and dietary monoamines such as serotonin, tyramine, and phenylethylamine. Here we show that MAO B, but not MAO A, gene expression was induced during Caco-2 cell differentiation; thus this cell line was used as a model system to study the gene regulation unique for MAO B. Luciferase and gel shift assays showed that transcription factors Sp1 and Sp3 binding to -246 and -99 bp were responsible for the observed gene activation. Overexpression of Sp3 inhibited the induction of MAO B gene by Sp1, and the expression of Sp3 was decreased during Caco-2 cell differentiation. Computer analysis revealed a putative CpG island containing 22 potential CpG methylation sites between -261 and -58 bp. In vitro methylation of MAO B promoter with 5-aza-2'-deoxycytidine, a DNA methyltransferase inhibitor, up-regulated MAO B gene expression in both HeLa and Caco-2 cells. Sodium bisulfite sequencing showed a gradually reduced methylation of the CpG sites during Caco-2 cell differentiation. These results suggested that MAO B gene expression is selectively induced by a decreased Sp3/Sp1 ratio and reduced DNA methylation. This new information may provide insights on the tissue-specific expression of these two isoenzymes.