Background:N-linoleyltyrosine (NITyr), one of the anandamide analogs, exerts activity via the endocannabinoid receptors (CB1and CB2), which showed anti-tumor effects in various tumors. Therefore, we speculated that NITyr might show anti-non-small cell lung cancer (NSCLC) effects via the CB1or CB2receptor. The purpose of the investigation was to reveal the anti-tumor ability of NITyr on A549 cells and its mechanisms.Methods:The viability of A549 cells was measured by MTT assay, and the cell cycle and apoptosis were both examined by flow cytometry; in addition, cell migration was tested by wound healing assay. Apoptosis-related markers were measured by immunofluorescence. The downstream signaling pathways (PI3K, ERK, and JNK) of CB1or CB2were examined through Western blotting. The expressions of CB1and CB2were detected by immunofluorescence. Finally, the AutoDock software was used to validate the binding affinity between the targets, such as CB1and CB2,with NITyr.Results:We found that NITyr inhibited cell viability, hindered the cell cycle, resulted in apoptosis, and inhibited migration. The CB1inhibitor, AM251, and the CB2inhibitor, AM630, weakened the aforementioned phenomenon. The immunofluorescence assay suggested that NITyr upregulated the expression of CB1and CB2. Western blot analysis indicated that NITyr upregulated the expression of p-ERK, downregulated the expression of p-PI3K, and did not affect p-JNK expression. In conclusion, NITyr showed a role in inhibiting NSCLC through the activation of CB1and CB2receptors involved in PI3K and ERK pathways.