The present study aimed to investigate the expression patterns of prothymosin-α (PTMA) and parathymosin (PTMS) in patients with squamous cell carcinoma (SCC), adenosquamous cell carcinoma (ASC) and adenocarcinoma (AC) of the gallbladder, and to assess their association with the clinicopathological characteristics and prognosis of the patients. A retrospective analysis of data pertaining to patients with SCC/ASC (n= 46) and AC (n= 80) of the gallbladder, who were treated with surgical resection, was conducted. Kaplan‑Meier survival analysis was also performed to assess the correlation of the expression pattern with survival. The results revealed a higher percentage of patients with a large tumor diameter (> 3 cm) in the SCC/ASC group as compared with those in the AC group (P< 0.05). No significant differences were observed between patients with SCC/ASC and those with AC with respect to the patient sex, presence of gallstones, TNM stage, lymph node metastasis, invasive growth into anatomically contiguous structures, surgical methods used, survival rate, and the expression levels of PTMA and PTMA (P> 0.05). However, positive expression of PTMA and PTMA was associated with tumor size, TNM stage, lymph node metastasis, locally invasive growth, and treatment with radical resection in patients with SCC/ASC and AC (P< 0.05). In addition, positive expression of PTMA and PTMA was observed in a significantly lower number of patients with advanced AC as compared with those in early AC (P< 0.05), while these expression levels were also associated with shorter survival in the SCC/ASC group and AC group (P< 0.05). Cox multivariate analysis also demonstrated a negative correlation between PTMA and PTMA levels, and the postoperative survival rate in the two groups. In conclusion, the present study indicated that the expression levels of PTMA and PTMA were closely associated with the tumorigenesis and progression of SCC, ASC and AC of the gallbladder. Positive expression of PTMA and PTMA may serve as a valuable prognostic factor in these patients.