The transcription factor YB‐1 is expressed in a wide range of cell types and has been implicated in the regulation of various genes involved in cell proliferation. Nuclear expression of YB‐1 is correlated with MDR‐1 gene expression in breast cancer and osteosarcoma. In this study, we asked whether YB‐1 expression is enhanced in human colorectral carcinoma and if it is associated with the expression of target genes such as MDR‐1, DNA topoisomerase II α and PCNA. YB‐1, DNA topoisomerase II α, PCNA and MDR‐1 expression were assessed by Western blotting, Northern blotting and immunohistochemistry in 26 human colorectal carcinomas. The involvement of YB‐1 in DNA topoisomerase II α gene expression was examined by transient DNA transfection assays. YB‐1 was overexpressed in almost all cancerous lesions in comparison with normal mucosa in surgically resected colorectal carcinomas of 26 patients. YB‐1 expression correlated well with both DNA topoisomerase II α and PCNA expression. In contrast, no correlation was observed between YB‐1 and MDR‐1 expression. We also found that a transient co‐transfection with a DNA topoisomerase II α promoter‐luciferase plasmid and an antisense YB‐1 expression construct resulted in a significant reduction of the promoter activity in KM12C human colon cancer cells. YB‐1 may be an excellent proliferation‐associated marker and may be a transcription factor regulating DNA topoisomerase II α gene expression in human colorectal carcinoma. Int. J. Cancer 83:732–737, 1999. © 1999 Wiley‐Liss, Inc.