Gallbladder cancer is a common malignancy among women in India. The aetiology of gallbladder carcinoma is still obscure and although numerous factors have been implicated, none has stood the test of time. This study was carried out to evaluate the role of lipid peroxidation product 4‐hydroxynonenal (HNE) in gallbladder carcinogenesis. High‐performance liquid chromatography (HPLC) analysis was carried out in 30 bile samples obtained during laparotomy from patients with gallbladder cancer (n = 10), cholelithiasis (n = 10) and age‐ and sex‐matched controls (n = 10). A significantly high concentration of HNE and lower concentration of biliary pH and electrolyte was observed in patients with carcinoma of the gallbladder compared with controls and patients with cholelithiasis. A significant correlation was also observed between biliary Na+and K+(r = 0.72), biliary Ca2+and Na+(r = 0.69) among controls. However, there was no correlation between HNE, electrolytes and pH among cancer patients. An increased concentration of biliary HNE and decreased concentration of electrolytes suggest biliary stasis rather then increased peroxidation in cancer patients.