Gallbladder carcinoma (GBC) possesses a poor prognosis, which is primarily attributed to the lack of early and timely surgical intervention. Calpain-1 and glypican-3 have been implicated in the progression of various types of cancer. The present study aimed to detect the expression of calpain-1 and glypican-3 in GBC, and analyzed whether the expression levels of these proteins correlated with any clinicopathological variables. A total of 100 patients with GBC and 30 patients with cholecystitis who accepted surgical treatment were enrolled in the present study. Pathological and clinical data were obtained from all patients. The expression of calpain-1 and glypican-3 was detected in paraffin-embedded tissues by immunohistochemistry. Calpain-1 expression was manually assessed with an immunohistochemical H-score with a slight modification. Glypican-3 expression was assessed as negative and positive. The correlations between protein expression and clinicopathological characteristics, and the associations between the proteins were analyzed. All patients exhibited positive expression of calpain-1. Notably, the high expression rate of calpain-1 was significantly increased in patients with GBC, compared with patients with cholecystitis (32.0 vs. 6.7%; χ2=7.668; P=0.006), suggesting that calpain-1 expression may be associated with progression from cholecystitis to GBC. In addition, the positive rate of glypican-3 expression was 53.0% in patients with GBC and 63.3% in patients with cholecystitis, with no significant difference (χ2=0.997; P=0.318). Furthermore, the expression of calpain-1 and glypican-3 had no significant correlation with gender, age, degree of tumor differentiation and tumor-node-metastasis classification in patients with GBC. Notably, the expression of calpain-1 and glypican-3 displayed a significant positive correlation in patients with GBC (r=0.517; P<0.01), but a significantly negative correlation (r=-0.856; P<0.01) in patients with cholecystitis. In conclusion, calpain-1 expression may be associated with progression from cholecystitis to GBC. Combined detection of calpain-1 and glypican-3 may be beneficial for prognosis assessment of GBC.