Abstract Introduction. Tumor metastasis is a primary cause for failure of locoregional therapy in colorectal cancer. Increased expression of osteopontin (OPN), a secreted phosphoprotein that binds to αvβ3 integrin and CD44 receptors, is associated with metastasis in several types of cancers. However, the mechanism by which OPN mediates metastasis is unknown. We hypothesized that OPN mediates interactions between tumor cells and extracellular matrix (ECM) to enhance metastasis. Methods. Using CT26 murine colon cancer cells, we generated a cell line (si-OPN) in which OPN expression was inhibited through stable transfection of small interfering RNA (siRNA) plasmids. Northern and Western blotting quantified OPN mRNA and protein levels. Wild-type (WT) and CT26 cells expressing mismatch-negative siRNA (si-Neg) served as controls. Matrigel/ECM migration, invasion, and adhesion assays and a murine hepatic-metastasis model were used to evaluate in vitro and in vivo metastatic potential amongst cell lines. Results. si-OPN cells demonstrated a 3.2-fold decrease in OPN protein expression and a 2.4-fold decrease in OPN mRNA expression in comparison to WT. OPN mRNA and protein levels in WT and si-Neg controls did not differ. In vitro assays demonstrated that cell motility and invasiveness were decreased in si-OPN by 2.0-fold ( P = 0.001 versus WT) and 2.5-fold ( P = 0.03 versus WT), respectively, but similar between WT and si-Neg controls. Cell adhesion was not different among cell lines. Intrasplenic injection of cells produced hepatic metastases in 33 mice (11/group). In vivo tumor burden was assessed by measuring liver weights. si-OPN cells demonstrated a 49.6% decrease in mean liver weight compared to WT (3.79 ± 0.49 g versus 1.88 ± 0.40 g, P < 0.05). Mean liver weights were similar between WT and si-Neg controls (3.79 ± 0.49 g versus 3.86 ± 0.43 g). Conclusion. RNA interference stably reduces CT26 tumor expression of OPN and significantly attenuates colon cancer metastasis in vivo. These data indicate that OPN mediates tumor metastasis by enhancing tumor cell invasion and migration through ECM.