Introduction: Osteopontin (OPN) is a glycosylated phosphoprotein found in the extracellular matrix that is expressed in a variety of body tissues and is involved in a variety of pathobiological processes such as cancer and metastasis. Several studies have reported increased OPN gene expression in advanced stages of breast cancer, but no studies have been conducted to investigate the relationship between OPN and metastasis in breast cancer. Materials and Methods: After the development of an animal model of breast cancer using the 4T1 cell line, primary and metastatic tumor cells were isolated from the tumor mass, lung, and brain of cancerous mice, multiplied, and named 4T1T, 4T1L, and 4T1B, respectively. The expression of OPN in these cells has been analyzed using real-time polymerase chain reaction. Results: According to our findings, metastatic tumor cells significantly increase their OPN expression. OPN expression was increased 2.3-fold and 3.25-fold in 4T1L and 4T1B cells, respectively, when compared to primary tumor cells. Conclusion: These findings provided important insights into OPN expression in the metastatic cascade of breast cancer for the first time. In this account, the analysis of the molecular properties of metastatic tumor cells can help researchers better understand the molecular and genetic aspects of chemoresistance, as well as design targeted therapeutic strategies to combat breast cancer metastasis.