In GBC, promoter hypermethylation occurs as an early event and can be a frequent mechanism for downregulation of BNIP3.

• Epigenetic changes have been documented as one of the risk factors of gallbladder cancer, and there exists an association of promoter hypermethylation with high incidence and poor prognosis of GBC.

• Bcl-2/adenovirus E1B 19 kDa interacting protein 3 is a pro-apoptotic protein member of Bcl-2 family.

• In this study of 84 GBC patients and 29 gallstone tissues (used as normal controls), down regulation of BNIP3 was evident in 56% of the GBC samples.

• Hypermethylation of BNIP3 promoter was also frequent (69%) in GBC samples.

• Reduced expression of BNIP3 was identified in 69% (40/58) of the BNIP3 promoter hypermethylated samples.

• Significant correlation of the mRNA expression and promoter hypermethylation with late stage and nodal metastasis was found.

• Hypermethylation of BNIP3 promoter was identified to be linked with low overall survival period.