Prostate cancer (PCa) is a multifactorial disease, which arises from the activation of genes related to cell proliferation and DNA synthesis. Two important genes namely hepsin (HPN) and inosine 5-monophosphate dehydrogenase type 2 (IMPDH2) were studied to understand the pathogenesis of this disease. This study aimed to determine the transcript level of PCa-related genes, HPN and IMPDH2, in archived tissues. Their functional roles were further determined using an in vitro model of PCa. Total RNA extraction was done from formalin-fixed paraffin-embedded PCa tissues, and benign prostatic hyperplasia (BPH) tissues acted as the control. Quantitative real-time polymerase chain reaction (qPCR) was performed to measure HPN and IMPDH2 expression. The functional assay was performed in a prostate cancer cell line (DU145) on these two genes by silencing their RNA. We discovered a significantly higher expression of IMPDH2 in PCa samples compared to non-cancerous tissues (P < 0.001). While HPN expression level showed a downward trend in PCa but the result was not statistically significant compared to the control. SiRNA-mediated knockdown of IMPDH2 expression in the cell line significantly decreased cell proliferation. The silencing of IMPDH2, however, did not affect cell migration, invasion, and apoptosis of the DU145 cell line. Our study demonstrated that IMPDH2 plays an essential role in clinical samples as well as in vitro models of this cancer. Inhibition of this gene through siRNA causes retardation of cell proliferation suggesting that IMPDH2 plays an essential role in prostate cancer.