Semaphorin family belongs to secreted or membrane anchored proteins. Recent studies have demonstrated the involvement of semaphorins and their receptors in cancer biology, but it remains unclear how each semaphorin molecule regulates tumorigenesis. Previously we reported that Semaphorin 3A (Sema3A) and its receptor Plexin A1 (PlxnA1) regulate the malignant phenotypes of mouse-derived lewis lung cancer (LLC) cells constitutively expressing GFP (LLC-GFP). Here we show that Semaphorin 6B (Sema6b) serves as one of the oncogenic semaphorin molecules in lung cancer using LLC-GFP. Sema3a or Plxna1 knockdown downregulated Sema6b, and their suppressive effect on proliferation was significantly recovered by recombinant SEMA6B (rSEMA6B) treatment. Sema6b knockdown suppressed the proliferation and tumorigenicity of LLC-GFP. Interestingly, the self-renewal capacity of LLC-GFP derived cancer stem-like cells (LLC-GFPstem) was completely lost by Sema6b knockdown. These results demonstrate that Sema6B would be the novel therapeutic target of lung cancer.