Tumor cell invasion and metastasis are hallmarks of malignancy. Despite recent advances in the understanding of lymphatic spread, the mechanisms by which tumors metastasize to sentinel/distant lymph nodes and beyond are poorly understood. To gain new insights into this complex process, we established highly metastatic melanoma cell lines by in vivo passaging the B16 parental cell line through the lymphatic system. In this study we characterized morphology, rate of cell proliferation, colony formation, migration, tumorigenicity, lymph flow, and capacities to induce tumor- and sentinel lymph node-lymphangiogenesis. Furthermore, microarray-based comparative analysis between parental and passaged cell lines was performed to identify specific gene expression profiles. The most differentially expressed gene was SPP (osteopontin), a secreted glycophosphoprotein which is known to be involved in cancer metastasis. Overexpression of osteopontin in B16 F1-variant was confirmed by western blot analysis and quantitative RT-PCR. Treatment of cultured lymphatic endothelial cells (LECs) with osteopontin promoted cell migration mediated by the integrin α9 pathway. Our results identify osteopontin as a novel lymphangiogenic factor.