Basic transcription factor 3 (BTF3) is associated with the development of several cancers. The aim of our study was to elucidate the role of BTF3 in colorectal cancer (CRC) tissues. CRC tissues or their paired adjacent noncancerous (ANCT) tissues were obtained from 90 patients who underwent operations in our hospital from November 2011 to December 2016, and then we implemented a gene microarray assay for detecting significant changes in gene expression and confirmed expression in tissues using immunohistochemistry and real-time PCR. We transfected or injected the silencing BTF3 (BTF3-siRNA) plasmid into cells and nude mice, and measured the tumorigenicity of CRC cells with flow cytometry and studied the expression level of BTF3 downstream genes (MAD2L2, MCM3 and PLK1) in CRC cells. BTF3 expression level was not only significantly higher in CRC tissue than in ANCT tissue (2.61 ± 0.07 vs 1.90 ± 0.03, P < 0.001) but BTF3-siRNA decreased tumor formation in a nude mice model. Furthermore, based on the data of gene microarray analysis, MAD2L2, MCM3 and PLK1 were detected as the downstream target genes of BTF3 and their expressions were positive related with BTF3 expression. Also, through transfecting BTF3-siRNA into HCT116 cells, we found that BTF3-siRNA could decrease cell viability and induced cell apoptosis and blocking the cell cycle. In conclusion, BTF3 is positively related to CRC and BTF3-siRNA attenuated the tumorigenicity of colorectal cancer cells via MAD2L2, MCM3 and PLK1 activity reduction.