The homeoboxB9 (HOXB9) gene is necessary for specification of the anterior-posterior body axis during embryonic development and expressed in various types of cancer. Here we show that the Wilms tumor transcription factor WT1 regulates the HOXB9 gene in a bidirectional manner. Silencing of WT1 activates HOXB9 in Wt1 expressing renal cell adenocarcinoma-derived 786-0 cells, mesonephric M15 cells and ex vivo cultured murine embryonic kidneys. In contrast, HOXB9 expression in U2OS osteosarcoma and human embryonic kidney (HEK) 293 cells, which lack endogenous WT1, is enhanced by overexpression of WT1. Consistently, Hoxb9 promoter activity is stimulated by WT1 in transiently transfected U2OS and HEK293 cells, but inhibited in M15 cells with CRISPR/Cas9-mediated Wt1 deletion. Electrophoretic mobility shift assay and chromatin immunoprecipitation demonstrate binding of WT1 to the HOXB9 promoter in WT1-overexpressing U2OS cells and M15 cells. BASP1, a transcriptional co-repressor of WT1, is associated with the HOXB9 promoter in the chromatin of these cell lines. Co-transfection of U2OS and HEK293 cells with BASP1 plus WT1 prevents the stimulatory effect of WT1 on the HOXB9 promoter. Our findings identify HOXB9 as a novel downstream target gene of WT1. Depending on the endogenous expression of WT1, forced changes in WT1 can either stimulate or repress HOXB9, and the inhibitory effect of WT1 on transcription of HOXB9 involves BASP1. Consistent with inhibition of Hoxb9 expression by WT1, both transcripts are distributed in an almost non-overlapping pattern in embryonic mouse kidneys. Regulation of HOXB9 expression by WT1 might become relevant during kidney development and cancer progression.