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BACKGROUND Little is known about cytochrome P450 3A4 (CYP3A4) DNA methylation and transcription alterations in gastric cancer. OBJECTIVE In this paper, we initially aimed to address the effect of diazinon pesticide on DNA methylation and transcription changes of the CYP3A4 gene in a human gastric cell line. In the next step, we studied the methylation differences of CpG sites within the upstream regulatory regions of the CYP3A4 gene among human gastric cancerous and healthy tissues. METHODS For the in vitro assay, the methylation changes of the C/EBP response element and transcript level of the CYP3A4 gene were studied following treatment of the AGS cell line with various concentrations of diazinon pesticide. In the next phase, the methylation percentages of 24 CpG sites within or around the upstream regulatory elements including near promoter, C/EBP binding site, XREM, and CLEM4 in 11 specimens of human gastric cancer tissue were compared to their adjacent healthy tissues. RESULTS Treatment with 10 µM Diazinon significantly increased the CYP3A4 gene transcription by approximately 27-fold, which was correlated with the hypermethylation of 3 CpGs in C/EBP binding sites including -5998, -5731 and -5725 (p<0.001 for all comparisons). Results of bisulfite sequencing revealed that the CpG sites which are located in -1521 (p=0.003), -1569 (p=0.027), -10813 (p=0.003), -10851 (p=0.001) and -10895 (p=0.0) bp from transcription start site, were significantly hypermethylated in cancerous tissues comparing to their healthy cohort. CONCLUSION Hypermethylation of CLEM4 and a region near the core promoter may have a significant association with gastric cancer incidence.