Bacillus Calmette‑Guérin (BCG) has become a significant treatment for bladder cancer, and neutrophils are reported to be associated with the antitumor effect of BCG. The aim of the present study was to clarify the antitumor function of neutrophils stimulated by BCG. Initially, the killing effect and cytotoxic activity of neutrophils treated with BCG was detected. Subsequently, the effectiveness of BCG‑treated neutrophils extracted from tumor‑bearing mice was analyzed. The results revealed that the cytotoxic effect of neutrophils was stronger in the BCG‑treated group compared with that in the normal saline (NS)‑treated and control groups (P<0.05). A significantly higher concentration of cytokines tumor necrosis factor (TNF)‑α, interleukin (IL)‑1β, IL‑6 and TNF‑related apoptosis‑inducing ligand occurred in the BCG‑treated neutrophil group compared with the NS and control groups (P<0.01), which was also associated with the BCG dose (P<0.01). The gross tumor volume percentage in BCG‑treated neutrophils from tumor‑bearing mice (BCGTgroup) was significantly lower in comparison with that in the NS‑treated neutrophils from tumor‑bearing mice (NSTgroup; P<0.05). In addition, the survival rate of tumor‑bearing mice was higher in the BCGTgroup compared with the NSTgroup (P<0.05), while more BCG‑treated neutrophils from tumor‑bearing mice were infiltrated in the MethA tumor (P<0.01). In conclusion, BCG‑treated neutrophils were observed to enhance the antitumor efficacy and extend the life span of mice.