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Prostate cancer (PCa) has high mortality rates, with most of the deaths resulting from the development of metastasis. Fibronectin (FN) plays key roles in cell adhesion and affects the migratory behavior of cells. In the tumor microenvironment and also in the blood plasma during metastasis, FN displays increased expression, however its role in prostate cancer remains poorly understood. This study aimed to unveil the specific roles of FN as a soluble component, alone or in combination with a complex basement membrane. To investigate the impact of FN in neoplastic prostate cells, we evaluated the gene expression of LNCaP cells by RT-qPCR after exposure to soluble FN (25 µg/mL) either alone or in combination with a basement membrane. When FN was the predominant matrix element, such as in blood plasma, PCa tumor cells increased their expression of genes related to an invasive behavior and resistance to apoptosis, including CDH2, ITGA5, AKT1, and BCL2. However, the combined presence of FN and a complex basement membrane had the opposite effect on LNCaP cells, in which the expression levels of CDH2, ITGA5, AKT1, and BCL2 were reduced. Hierarchical clustering analysis with LNCaP and RWPE-1 cells showed that LNCaP cells exposed to an enriched extracellular matrix displayed an expression pattern more similar to that shown by RWPE-1 cells, a cell line that illustrates characteristics of the normal prostate epithelium. These findings provide the groundwork for future studies addressing the role of FN in tumor growth, particularly in the context of cancer evolution/progression from a solid primary tumor to a transitory circulating state.