Previous studies from our laboratory have identified several endothelial cell-associated marker genes implicated in human melanoma metastasis via tumor vasculogenic mimicry. In this study, we used dual model systems composed of melanoma cell lines and clinical melanoma samples to validate the importance of insulin-like growth factor binding protein-3 (IGFBP-3) as a marker involved in disease progression. Gene expression analysis was done using a microarray approach for both primary and metastatic melanoma samples. The expression of IGFBP-3 was decreased using a small interfering RNA (siRNA) knockdown approach and quantified with real-time quantitative reverse transcription-PCR analysis. The expression of insulin-like growth factor binding protein 3 (IGFBP-3) was up-regulated by nearly 16-fold in WM266-4 compared with WM35 cells. A subsequent parallel analysis using freshly isolated primary and metastatic melanoma cell samples and melanoma tissue array confirmed the previous findings. The functional significance of IGFBP-3 in melanoma invasion was further investigated using a siRNA gene knockdown approach, with the expression of IGFBP-3 markedly reduced. Additionally, siRNA knockdown resulted in a significant reduction in cell motility, migration, and invasive capacity of WM266-4 cells in vitro. These results strongly suggest that IGFBP-3 expression may be a vital cell motility, migration, and proliferation factor necessary for melanoma metastasis and is an important biomarker in human melanoma.