The Glycyrrhiza uralensis Fisch, one of the famous Chinese medicinal herbs, has been widely used in anti-cancer prescription. We found that glycerrhetinic acid (GA, triterpenoid) and licochalcone A (LCA, flavonoid), two primary constituents of Glycyrrhiza uralensis Fisch, induced cell proliferative inhibition, apoptosis, and autophagy in non-small cell lung cancer (NSCLC) A549 and NCI-H1299 cells. Combined treatment with chloroquine further enhanced GA- or LCA-induced expression of LC3-II and more red-fluorescent puncta than green ones were observed in GA- or LCA-treated cells with transfection of mRFP-EGFP-LC3, suggesting that GA and LCA induces autophagic flux in NSCLC cells. Inhibition of autophagy enhanced GA-induced cell proliferative inhibition and apoptosis in NSCLC cells. In constant, LCA-induced cell proliferative inhibition and apoptosis were not obviously changed after autophagy inhibition in NSCLC cells. The JNK and IRE1α were activated after incubation with GA. Pretreatment with the JNK inhibitor SP600125 or silencing of the JNK pathway by siRNA of JNK or c-jun decreased GA-induced autophagy. Moreover, the GA-induced JNK pathway activation and autophagy were decreased by IRE1α knockdown, suggesting that GA induces autophagy through activation of IRE1α-JNK/c-jun pathway. LCA increased the expression of CHOP, and knockdown of CHOP reversed LCA-induced autophagy, suggesting that LCA-induced autophagy is due to induction of CHOP expression. Collectively, both GA and LCA induced cell proliferative inhibition, apoptosis, and autophagy in NSCLC, and inhibition of autophagy might be an effective strategy to enhance the anti-NSCLC effects of Glycyrrhiza uralensis Fisch contained prescriptions.