Exploring key genes associated with non-small cell lung carcinoma (NSCLC) may lead to targeted therapies for NSCLC patients. The protein kinase MAP4K3 has been established as an important modulator of cell growth and autophagy in mammals. Herein, we investigated the somatic mutations and the expression pattern of MAP4K3 detected in NSCLC patients based on the TCGA database. Abnormal MAP4K3 expression and its somatic mutations are associated with the carcinogenesis and thereby becoming an attractive therapeutic target. Baicalein, a natural product, was determined to be the first-reported MAP4K3 binding ligand with its K_D values of 6.47 μM measured by microscale thermophoresis. Subsequent in silico docking and mutation studies demonstrated that baicalein directly binds to MAP4K3, presumably to the substrate-binding pocket of this kinase domain, causing inactivity of MAP4K3. We further showed that baicalein could induce degradation of MAP4K3 through decreasing its stability and promoting the ubiquitin proteasome pathway. Degradation of MAP4K3 could cause dissociation of the transcription factor EB and 14-3-3 complex, enhance rapid transport of TFEB to the nucleus and trigger TFEB-dependent autophagy, resulting in lung cancer cells proliferation arrest. Knockdown of MAP4K3 expression by siRNA was sufficient to mimic baicalein-induced autophagy. Ectopic expression of the MAP4K3 protein resulted in significant resistance to baicalein-induced autophagy. Baicalein exhibited good tumor growth inhibition in a nude mouse model for human H1299 xenografts, which might be tightly related to its binding to MAP4K3 and degradation of MAP4K3. Our data provide novel mechanistic insights of baicalein/ MAP4K3/ mTORC1/ TFEB axis in regulating baicalein-induced autophagy in NSCLC, suggesting potential therapies for treatment of NSCLC.