Tumor-initiating cells possess the capacity for self-renewal and to create heterogeneous cell lineages within a tumor. Therefore, the identification and isolation of cancer stem cells is an essential step in the analysis of their biology. The aim of the present study was to determine whether the cell surface protein neuropilin 1 (NRP1) can be used as a biomarker of stem-like cells in lung cancer tumors. For this purpose, NRP1-negative (NRP1-) and NRP1-positive (NRP1+) cell subpopulations from two lung cancer cell lines were sorted by flow cytometry. The NRP1+ cell subpopulation showed an increased expression of pluripotency markers OCT-4, Bmi-1 and NANOG, as well as higher cell migration, clonogenic and self-renewal capacities. NRP1 gene knockdown resulted not only in a decreased expression of stemness markers but also in a decrease in the clonogenic, cell migration and self-renewal potential. In addition, the NRP1+ cell subpopulation exhibited dysregulated expression of epithelial-to-mesenchymal transition-associated genes, including the ΔNp63 isoform protein, a previously reported characteristic of cancer stem cells. Notably, a genome-wide expression analysis of NRP1-knockdown cells revealed a potential new NRP1 pathway involving OLFML3 and genes associated with mitochondrial function. In conclusion, we demonstrated that NRP1+ lung cancer cells have tumor-initiating properties. NRP1 could be a useful biomarker for tumor-initiating cells in lung cancer tumors.