Myricetin is a typical flavonol with various pharmacological effects which shows favorable biological activities in cancer. However, the underlying mechanisms and potential targets of myricetin in NSCLC (non-small cell lung cancer) cells remain unclear. First, we demonstrated that myricetin not only inhibited the proliferation, migration and invasion, but also induced apoptosis in A549 and H1299 cells in a dose-dependent manner. Then, we confirmed myricetin may play an anti-NSCLC effect through modulating MAPK-related functions and signaling pathway by Network pharmacology. Furthermore, MKK3 (MAP Kinase Kinase 3) was identified and confirmed as a potential target of myricetin by biolayer interferometry (BLI) and molecular docking, revealing that myricetin directly bound to MKK3. Moreover, three mutations (D208, L240, and Y245) of key amino acids predicted by molecular docking obviously decreased the affinity between myricetin and MKK3. Finally, enzyme activity assay was utilized to determine the effect of myricetin on MKK3 activity in vitro, and the result showed that myricetin attenuated MKK3 activity. Subsequently, myricetin decreased the phosphorylation of p38 MAPK. Furthermore, knockdown of MKK3 reduced the susceptibility of A549 and H1299 cells to myricetin. These results suggested that myricetin inhibited the growth of NSCLC cells via targeting MKK3 and influencing the downstream p38 MAPK signaling pathway. The findings revealed that MKK3 is a potential target of myricetin in the NSCLC and myricetin is considered to be a small-molecular inhibitor of MKK3, which can improve comprehension of the molecular mechanisms of myricetin pharmacological effects in cancer and further development of MKK3 inhibitors.