Ganglioside GM3 has recently been shown to regulate tumor necrosis factor (TNF)-α at both the transcriptional and translational levels in murine melanoma B16 cells (Wang et al., Biochem Biophy Res Commun 356:438-443, 2007; Wang et al., Oncology 73:430-438, 2007). Since TNF-α is known to stimulate matrix metalloproteinase (MMP)-9 synthesis, which is highly involved in tumor cell metastasis, we considered whether MMP-9 is regulated by GM3. Expression of GM3 in B16 cells was modified by genetic manipulation, exogenous GM3 addition, and inhibition of GM3 synthesis followed by determination of MMP-9 expression by reverse transcriptase-polymerase chain reaction (RT-PCR) and/or gelatin zymography. We determined that MMP-9, but not MMP-2, expression is consistent with GM3 levels in several B16 cell variants produced by genetic manipulation, while MMP-9 is increased by GM3 addition to these cells and decreased with inhibition of glycolipid synthesis by d-threo-1-phenyl-2-decanoylamino-3- morpholino-1-propanol (D-PDMP). GM3 stimulation of cells enhanced AKT phosphorylation as well as MMP-9 synthesis. LY294002 showed a potent inhibitory effect on MMP-9 synthesis and AKT phosphorylation at Ser 473, either with or without GM3 stimulation, indicating a central role for the PI3K/AKT pathway in this process. An MMP inhibitor, GM6001, clearly inhibited cell motility by suppressing MMP-9 expression and activation. Taken together, these results support the notion that the ganglioside GM3 positively regulates MMP-9 expression (but not MMP-2) and may contribute to murine melanoma metastasis via activation of MMP-9 through the PI3K pathway.