Cancer associated fibroblasts (CAFs) significantly influence the proliferation, invasion, and metastasis of cancer cells. CAFs are detected in most tumors and provide a ubiquitous target that is being actively investigated in cancer treatment. In prostate cancer, fibroblasts have been shown to induce growth, confer castration-resistance, and increase metastatic potential. Choline kinase (Chk)-α downregulation has been previously shown to significantly decrease cancer cell viability but its effect on CAFs has not been investigated before. Here we found, for the first time, a significant decrease of prostate cancer fibroblast (PCAF) viability with Chk-α downregulation.