BACKGROUND: Lung cancer is the leading cause of cancer-related death worldwide, of which anaplastic lymphoma kinase fusion positive (ALK (+)) non-small cell lung cancer (NSCLC) accounts for 3-7. Here, we identified a new fusion gene PLCXD3-ALK (P1, A19) from a patient with advanced lung squamous cell carcinoma (LUSC) by next-generation sequencing (NGS). We aimed to evaluate its oncogenic potential by performing functional studies in vitro and tumorigenicity in vivo of this fusion protein. METHODS: We performed functional experiments in NIH-3T3 cells with stable expression of PLCXD3-ALK including soft agar colony formation assay, cell proliferation and viability assays, and transwell assay. The activation of downstream pathways and the response to ALK inhibitors crizotinib and alectinib were demonstrated by western blotting (WB). In addition, we further evaluated the tumorigenicity of the PLCXD3-ALK mutants in nude mice. RESULTS: Similar to EML4-ALK, the PLCXD3-ALK fusion promoted proliferation and the capacity for non-anchorage-dependent growth of NIH-3T3 cells. We demonstrated that PLCXD3-ALK can activate ALK self-phosphorylation and downstream pathways, which could be inhibited by the addition of ALK inhibitors. Moreover, we observed that this gene could provoke oncogenic transformation in nude mice. Meanwhile, the patient was monitored for disease progression with computed tomography (CT) scanning during treatment with alectinib, and a benefit was observed. CONCLUSIONS: We identified and functionally validated PLCXD3-ALK as a novel rare fusion in NSCLC that has not been previously reported. It can serve as a meaningful therapeutic target for ALK inhibitors of ALK (+) NSCLC.