Objective To investigate the effect of bacillus Calmette-Guérin (BCG) on bladder cancer cells and their metabolites, and to preliminarily explore the possible mechanisms of BCG in the treatment of bladder cancer. Methods The rat model of bladder cancer was induced by intravesical instillation with N-methylnitrosourea (MNU). Bladder cancer cells and normal transitional epithelial cells were isolated and primarily cultured, and were divided into 5 groups according to the different components of the culture medium. The concentration of tumor necrosis factor-α (TNF-α) and interleukin-10 (IL-10) in the supernatant of each group was detected by enzyme linked immunosorbent assay (ELISA). The concentration of BCG to inhibit the cancer cell growth was determined by MTT assay. Apoptosis of bladder cancer cells was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). Results Among the 15 rats, 2 rats died after 2 times of instillation, and 3 rats died after 3 times of instillation, without obvious tumors found at autopsy. The other 10 rats were killed after completion of the intravesically instillation of MNU, and obvious tumors were found in 8 of them after dissection. The results of MTT assay showed that BCG had an inhibitory effect on the growth of bladder cancer cells, and the inhibitory rate was positively correlated with the concentration of BCG. The results of ELISA showed that the concentrations of TNF-α in the supernatant of groups B and D were (160.654±5.775) ng/L and (124.443±4.972) ng/L, respectively, with significant differences from those of the other three groups. The concentrations of IL-10 in the groups B and E were (16.973±3.428) ng/L and (20.327±2.721) ng/L, significantly higher than those of the other three groups. Apoptosis of cancer cells was not found in all groups. HE staining of the primary bladder cancer cells showed that the volume of cell nucleus was increased, and the nucleo-cytoplasmic ratio was increased. The number of nucleoli in some cells was increased and some nuclei appeared like ink drops with prominent nucleoli. Conclusions BCG has an inhibitory effect on the growth of rat bladder cancer cells. IL-10 and TNF-α secreted by the tumor cells might be involved in this regulatory process. However, apoptosis does not show an obvious effect on this inhibitory process.