Anti-metastasis by reducing cellular migration and invasion and by deregulating the expression of matrix metalloproteinases (MMPs) is a therapeutic approach for cancer treatment. The objective of this study focused on the effects of the novel compound 6-pyrrolidinyl-2-(2-hydroxyphenyl)-4-quinazolinone (MJ-29) regarding anti-metastatic actions on human oral squamous cell carcinoma CAL 27 cells and on the verification of the underlying related molecular mechanisms of this event. MJ-29 concentration- and time-dependently caused a suppression of cell adhesive ability utilizing cell adhesion assay; it also inhibited the migration and invasion of CAL 27 cells using scratch wound closure and transwell invasion assays in a concentration-dependent response. Importantly, we confirmed that the applied concentration range of MJ-29 exhibited no dramatic influence of cytotoxicity on CAL 27 cells using the thiazolyl blue tetrazolium bromide assay. MJ-29 also attenuated the enzymatic activity of MMP-2 and MMP-9. Furthermore, we found that activation of their upstream protein kinases, by MJ-29, potentially exerted an inhibitory effect on the phosphorylated protein levels of extracellular regulated protein kinase 1/2, p38 and c-Jun N-terminal kinase 1/2, as well as serine/threonine kinase AKT by MJ-29 in CAL 27 cells. The expression of RAS and focal adhesion kinase was also down-regulated in MJ-29-treated CAL 27 cells. Collectively, these findings provide further evidence for the molecular signaling basis of the effects of MJ-29 on suppression of migration and invasion which might be useful as a therapeutic strategy to treat human oral cancer.