Our earlier study revealed that long-term ethidium bromide application causes mitochondrial DNA depletion in human prostate cancer DU145 cell line (DU145(MtDP)), and this DU145(MtDP) subline appears to have expanded CD44(Bright) cell population than its parental wild type DU145 cells (DU145(WT)). Increasing evidence suggests that CD44(Bright) cells are highly cancer stem cell like, but it is not clear about their dynamic transition between CD44(Dim) and CD44(Bright) phenotypes in prostate cancer cells, and how it is affected by mitochondrial DNA depletion. To address these questions, four cell subpopulations were isolated from both DU145(WT) and DU145(MtDP) cell lines based on their CD44 expression level and mitochondrial membrane potential. The cell motility and colony formation capability of the fluorescence activated cell sorting-sorted cell subpopulations were further examined. It was discovered in the DU145(WT) cells that CD44(Dim) cells could transit into both CD44(Dim) and CD44(Bright) phenotypes and that CD44(Bright) cells were prone to sustain their CD44(Bright) phenotype as renewal. However, such transition principle was altered in the DU145(MtDP) cells, in which CD44(Bright) cells showed similar capability to sustain a CD44(Bright) phenotype, while the transition of CD44(Dim) cells to CD44(Bright) were suppressed. It is concluded that mitochondrial DNA depletion in the human prostate cancer DU145 cells influences their renewal and CD44 subphenotype transition. Such alterations may be the driving force for the enrichment of CD44(Bright) DU145 cells after the mitochondrial DNA depletion, although the molecular mechanisms remain unclear.