Thrombospondin-1 (TSP-1) is a multifunctional matricellular glycoprotein involved in several mechanisms critical to the formation and progression of solid tumors including cell adhesion, proliferation, migration, invasion, and angiogenesis. However, work related to TSP-1 expression and functionality in prostate cancer is limited. Expression experiments in the present study demonstrated lower expression of TSP-1 in the prostate cancer cell lines DU 145 and LNCaP compared to SV40-immortalized prostatic epithelial cells PNT 1A. All three cell lines expressed the TSP-1 receptor CD36. Exogenously added TSP-1 modulated the cellular phenotype of LNCaP cells, which demonstrated decreased proliferation rate and partly entered apoptosis. Collectively, these data support the concept that partial or complete loss of TSP-1 synthesis may provide tumor cells with a proliferation advantage. In addition, TSP-1 located at the border between tumor and stroma as observed in primary prostate tumors may act as a barrier of tumor growth depending on the TSP-1 receptor repertoire of the tumor cells.