Androgen receptor (AR) signals play a decisive role in regulating the growth and differentiation of both normal and cancerous prostate cells by triggering the regulation of target genes, in a process in which AR cofactors have critical functions. Because of the highly prostate-specific expression pattern of HOXB13, we studied the role of this homeodomain protein in prostate cells. Expression of HOXB13 was limited to AR-expressing prostate cells. Reporter transcription assay demonstrated that HOXB13 significantly suppressed hormone-mediated AR activity in a dose-responsive manner, and suppression was specific to AR with which HOXB13 physically interacts. Overexpression of HOXB13 further down-regulated the androgen-stimulated expression of prostate-specific antigen, and suppression of endogenous HOXB13 stimulated transactivation of AR. Functionally, HOXB13 suppressed growth of LNCaP prostate cancer cells, which could be counteracted by additional hormone-activated AR. On the other hand, the growth-suppressive function of HOXB13 in AR-negative CV-1 cells was not affected by AR. These results suggest that HOXB13 functions as an AR repressor to modulate the complex AR signaling and subsequent growth regulation of prostate cancer cells. In addition to the loss of HOXB13 expression, maintaining AR may be an important step for prostate cancer cells to tolerate the suppressor function of HOXB13. Altogether, our data present a novel mechanism for the HOXB13-mediated repression of AR signaling, which can be interpreted to a growth-suppressive event.