The present study was designed to investigate roles for NF-kappa B and the endogenous H2S producing enzyme cystathionine-beta-synthetase (CBS) signaling pathways in adult rats with experimental diabetes. Here, we showed that injection of STZ produced gastric hypersensitivity in female rats in response to gastric balloon distention in association with upregulation of CBS and p65 expression in gastric DRGs. Treatment with CBS inhibitor AOAA attenuated gastric hypersensitivity. AOAA treatment also reversed hyperexcitability of gastric-specific DRG neurons in diabetic rats. Blockade of NF-kappa B signaling using PDTC reversed upregulation of CBS expression. STZ treatment led to a significant demethylation of CpG island in cbs gene promoter region determined by methylation specific PCR and bisulfite sequencing. STZ treatment significantly enhanced cbs ability to bind DNA at p65 consensus site by CHIP assays. Our findings suggest that upregulation of cbs expression is attributed to cbs promoter DNA demethylation and p65 activation and that the enhanced interaction of cbs gene and p65 would contribute to gastric hypersensitivity in diabetes.