© 2018 Xie et al. Background: Squamous non-small cell lung cancer (SqNSCLC), as a leading cause of cancer-related deaths worldwide, has limited treatment options and poor prognosis. Thus, novel targeted therapies are desperately needed. Materials and methods: SqNSCLC cases from derivation and validation cohorts were analyzed for podoplanin (PDPN) expression, and its clinicopathological correlation and prognostic prediction. The Human Proteome Map database was used to compare the expression of different lung cancer targets in normal human tissues. Two human lung cancer cell lines, H226 (a SqNSCLC line) and A549 (a non-SqNSCLC line), were examined for PDPN expression. The in vitro cytotoxicity of an anti-PDPN therapy (NZ-1-immunotoxin [NZ-1-IT]) was tested against both lines. The in vivo therapeutic effect of NZ-1-IT was examined in subcutaneous non-small cell lung cancer (NSCLC) xenograft mouse models. Results: In the derivation cohort, 40% (28/70) were PDPN positive. There was significantly increasing pleural invasion (46.4% vs 9.5%, p=0.001), lymphovascular invasion (25.0% vs 9.5%, p=0.08), and lymph node involvement (53.6% vs 33.3%, p=0.09) in PDPN-positive vs PDPN-negative patients, along with poorer progression-free survival in PDPN-positive patients (p=0.07). The validation cohort with 224 randomly matched cases from The Cancer Genome Atlas data set also displayed significantly shorter overall survival in the group with elevated PDPN mRNA (p=0.05). However, PDPN showed limited expression in normal tissues. PDPN was highly and specifically expressed on the surface of H226 cells instead of A549 cells. Subsequently, PDPN-positive H226 cells were around 800 times more sensitive to anti-PDPN NZ-1-IT therapy than PDPN-negative A549 cells in vitro. Furthermore, NZ-1-IT significantly delayed tumorigenesis only in the H226 subcutaneous mouse model (p<0.05). Conclusion: Our results demonstrate a distinctively elevated expression of PDPN in SqNSCLC, which is significantly associated with worse clinicopathological features and poorer prognosis. With promising preclinical therapeutic results, anti-PDPN targeted therapy can thus be a robust potential strategy for future SqNSCLC treatment.