Increasing evidence indicates that macrophages in tumor stroma can significantly modify the malignant phenotypes of tumors. Osteopontin (OPN) is frequently overexpressed in cancers with high metastatic capacity and, thus, has been considered as a potential therapeutic target. To find out whether macrophages can affect the outcome of OPN-knockdown tumor cells, we used RNA interference (RNAi) to stably silence the OPN expression in the highly invasive human hepatoma cell line SK-Hep-1. Silencing of OPN markedly decreased the motility and invasiveness of the SK-Hep-1 cells. Further studies using this cell model revealed that coculture with human macrophages or macrophage-conditioned medium largely restored the migration and invasion potential of OPN-knockdown tumor cells. Moreover, such macrophage-promoted motility can be effectively blocked either by the addition of OPN-neutralizing antibody to the cocultured medium or by silencing OPN expression in macrophages. These results indicate that macrophage-derived OPN can compensate for the decrease of OPN and thereby restore the metastatic potential of OPN-knockdown tumor cells. Further characterization of the underlying mechanisms disclosed that macrophage-derived OPN exerted its function independently of the actin cytoskeleton rearrangement or the activation of matrix metalloproteinase and Rho families. Our results suggest that there are fine-tuned complex interactions between cancer cells and stroma cells, which may modify the outcome of cancer therapy, and therefore should be considered for the rational design of anticancer strategy.