Objective To investigate the gene expression profile of CSCs and to explore the key pathways and specific molecular signatures involved in the characteristic of CSCs. Materials and methods CD133+ /CD44+ CSCs and bulk population (non-CSCs) were isolated from DU-145 cells using fluorescence-activated cell sorting (FACS). We used Illumina HumanHT-12 v4 Expression to investigate gene expression profiling of CSCs and non-CSCs. Protein-protein interaction (PPI) network analysis was performed using the STRING database. Biomarkers selected based on gene expression profiling were visually analyzed using immunofluorescence staining method. An image analysis program, ImageJ (R), was used for the analysis of fluorescence intensity. Results In microarray analysis, we found that many ribosomal proteins and translation initiation factors that constitute the mTOR complex were highly expressed. PPI analysis using the 33 genes demonstrated that there was a close interaction between ribosome biogenesis, translation, and mTOR signaling. The fluorescence amount of mTOR and MLST8 were higher in CSCs compared to non-CSCs. Conclusions The increase in a number of genes associated with ribosome biogenesis, translation, and mTOR signaling may be important to evaluate prognosis and determine treatment approach for prostate cancer (PCa). A better understanding of the molecular pathways associated with CSCs may be promising to develop targeted therapies to prolong survival in PCa.