〔Abstract〕 Objective To investigate the functions of LIM-domain protein 4(LMO4)in regulating CD133 expression and proliferation of prostate cancer cells and its mechanism. Methods Immunohistochemistry(IHC)was used to detect the expression of LMO4 and CD133 in prostate cancer tissues and prostatic hyperplasia tissues. Fluorescence activated cell sorting was used to harvest CD133+and CD133-cells. Colony formation assay was performed to determine proliferation ability. RNA interference was performed to knockdown Lmo4 expression in PC-3 cells.Western blot and immunofluorescence assay were used to detect the expression of LMO4 and CD133 protein in PC-3 cells or PC-3/Si Lmo4 cells. Flow cytometry tested the CD133+cells number and cells proliferation ability. Colony formation assays were used to determine PC-3 cells clonogenic capability. Co-Immunoprecipitation( Co-IP) was used to detect the interaction between LMO4 and cyclin-dependent kinase 9( CDK9). Results Comparing to prostatic hyperplasia tissues,CD133 and LMO4 expresssions dramatically increased in prostate cancer tissues. CD133+subpopulation of PC-3 cells exhibited high clonogenic capability. Both CD133 expression and numbers of CD133+cells significantly decreased in LMO4 knockdown PC-3 cells. Furthermore,silencing Lmo4 led to decrease the abilities of cell proliferation and clonogenic. Co-immunoprecipitation analysis showed that LMO4 and CDK9 could form a LMO4/CDK9 complex in prostate cells. However,CDK9 may leave from the complex owing to LMO4 overexpression in prostate cells. Interestingly,the LMO4/CDK9 complex was not detected in PC-3/Si Lmo4 cells. Conclusion Up-expression of the nuclear transcription factor LMO4 is consistent with higher expression of cancer stem marker CD133 in prostate cancer. Prostate cancer cells proliferation may be regulated by LMO4 in an independent-LMO4/CDK9 complex manner.