Metastatic cancer cells use the actin-bundling process to constantly remodel the actin cytoskeleton for adhesion, migration, and invasion. L-plastin is an actin-bundling protein that belongs to the plastin family. L-plastin has been identified in several malignant tumors of the colon, prostate, and breast and contributes to cancer cell invasion in a phosphorylation-dependent manner. Our initial characterization in PC3 prostate cancer (PCa) cell line derived from bone metastases demonstrated L-plastin expression in PCa cells but not in other PCa cell lines tested. Hence, this study aimed to identify L-plastin’s role in the migration and invasion of PC3 cells. Immunostaining analysis demonstrated a punctate distribution of L-plastin and patchy actin staining in PC3 cells with a minimal colocalization between L-plastin and actin at the invadopodia. However, L-plastin overexpression in PC3 cells increased L-plastin’s colocalization and actin at the invadopodia and during the invasion. In a wound-healing assay, these cells displayed a significant reduction in migration. L-plastin and invadopodia connections were confirmed using the L-plastin knockdown strategy in PC3 cells (PC3/Si). PC3/Si cells demonstrated an increased migration, which corresponded to punctate podosome-like structures. However, a decrease in the number of invadopodia contributed to a significant reduction in the invasion. Additionally, tumorsphere formation was significantly reduced in PC3/Si cells than in PC3 cells. In conclusion, our observations suggest that L-plastin regulates the formation of invadopodia required for prostate cancer invasion. Our results highlight that it could be a novel therapeutic target for androgen-independent metastatic prostate cancer.Up to 350 words. No references allowed. Abstracts may be submitted at a later date.