Bladder cancer is the most common malignant disease of the urinary tract and the ninth most common malignancy in the world. The risk factors for the development of this disease have not been sufficiently studied and are poorly supported by basic studies in vivo and in vitro. In this paper, we studied the influence of the microbiota and antibiotics on the metabolism of urothelial procarcinogen N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN) using a mouse model and a HepG2 cell line. The effect of antibiotics, on the hepatic metabolism of this compound, was studied on hepatocyte cell culture by exposing the cells to antibiotics and incubating them with BBN. The amount of carcinogenic metabolite formed was measured by high-performance liquid chromatography (HPLC). On a mouse model, we studied the effect of antibiotic microbiota reduction on gene expression, which is thought to participate in the transformation of BBN into its carcinogenic form, in liver and bladder tissue. Gene expression was measured by a quantitative polymerase chain reaction. In vivo experiments showed that the expression of four genes involved in BBN metabolism in the liver changed in response to microbiota reduction in mouse tissue. From the above, it can be concluded that, by altering gene expression, the microbiota can affect the metabolism of xenobiotics. In vitro experiments have found that antibiotics do not affect the ability of hepatocytes to metabolize BBN.