Recently, epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) have revolutionized non-small cell lung cancer (NSCLC) treatment. However, resistance remains a major obstacle. Anexelekto (AXL) is a member of receptor tyrosine kinases (RTKs) and shares the same downstream signaling pathways with EGFR, such as PI3K/AKT and MAPK/ERK. AXL overexpression in resistant tumors has been implicated in many previous studies in vitro and in vivo. In this study, we further examined whether expression of AXL and its downstream targets increased in gefitinib-resistant PC9 cells (PC9GR). In addition, we hypothesize that knocking down AXL in PC9GR and overexpressing AXL in PC9 using genetic tools can restore and decrease the sensitivity to gefitinib, respectively. We found that silencing AXL could sensitize the resistance to gefitinib, and the downstream pathways were significantly inhibited. Interestingly, we also discovered that increased AXL expression did promote the resistance, and its downstream targets were activated accordingly. Then 69 NSCLC patients who harbored EGFR mutation were recruited to analyze the expression of AXL and the association between AXL expression and clinical characteristics. We found that 5 of the 69 patients were AXL positive (about 7%), and AXL was related to tumor differentiation and tumor size. In this study, we concluded that the molecular mechanisms of AXL mediated resistance involved in the increased activity of the PI3K/AKT and MAPK/ERK1/2 pathways, and AXL overexpression could promote resistance, but it can be weakened when AXL expression is silenced.