GM3, the simplest ganglioside, regulates cell proliferation, migration, and invasion by influencing cell signaling at the membrane level. Although the classic N-acetylated form of GM3 (NeuAcLacCer) is commonly expressed and has been well studied, deacetylated GM3 (NeuNH2LacCer, d-GM3) has been poorly investigated, despite its presence in metastatic tumors but not in noninvasive melanomas or benign nevi. We have recently found that d-GM3 stimulates cell migration and invasion by activating urokinase plasminogen activator receptor (uPAR) signaling to augment matrix metalloproteinase-2 (MMP-2) function. However, the mechanisms by which d-GM3/uPAR increase MMP-2 expression and activation are not clear. By modifying the expression of d-GM3 genetically and biochemically, we found that decreasing d-GM3 expression inhibits, whereas overexpressing d-GM3 stimulates, p38 mitogen-activated protein kinase (MAPK) activity to influence MMP-2 expression and activation. p38 MAPK (p38) activation requires the formation of a membrane complex that contains uPAR, caveolin-1, and integrin alpha5beta1 in membrane lipid rafts. In addition, knocking down or inhibiting focal adhesion kinase (FAK), phosphoinositide 3-kinase (PI3K), or Src kinase significantly reduces d-GM3-induced p38 phosphorylation and activation. Taken together, these results suggest that d-GM3 enhances the metastatic phenotype by activating p38 signaling through uPAR/integrin signaling with FAK, PI3K, and Src kinase as intermediates. Elucidation of the mechanisms by which d-GM3, a newly discovered, potential biomarker of metastatic melanomas, promotes cell metastasis will help us to understand the function of d-GM3 in metastatic melanomas and may lead to novel GM3-based cancer therapies.