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The present study investigates the role of DNA methylation in regulation of deacetylvindoline-4-O-acetyltransferase (DAT) gene in Madagascar periwinkle (Catharanthus roseus). Expression analysis showed significant upregulated DAT transcript (20 fold), demeter like (1.37 fold) and repressor of silencing-1 (1.47 fold) in leaf tissues of C. roseus as compared to root tissues. Methylases such as domain rearranged methyltransferase and chromomethyl transferase were downregulated suggesting involvement of DNA methylation in DAT regulation. DNA methylation signatures on DAT gene were further evaluated after treatment of leaves from C.roseus with 5-azacytidine (20 μM) for 5 and 10 days. Promoter DNA (− 11 to − 378 region) methylation analysis showed hypermethylation on two CHG and three CHH sites after day 10 of treatment. Qualitative methylation specific PCR of the coding region (+ 153 to + 322 region) showed hypomethylation after 10 days of 5-azacytidine treatment. Expression analysis showed upregulated DAT gene expression in the leaves 10 days post treatment, which correlated with changes observed in DNA methylation. Moreover, genomic 5-methylcytosine levels decreased from 0.65 to 0.33% after 10 days of 5-azacytidine treatment. However, no significant change was observed after 5 day of 5-azacytidine treatment as compared to control across different observational end points. The study clearly suggests that DAT gene expression may be regulated by differential DNA methylation in the promoter and coding regions. Further, hypomethylating agent such as 5-azacytidine holds potential to regulate the expression of DAT that regulates the terminal step in the indole alkaloid pathway leading to vindoline synthesis.