Plexin (Plxn) family molecules are the receptor for semaphorins and their involvement in tumor malignancy is also demonstrated. However, the regulatory roles of Plxns in cancer-stemness have not been completely understood. Previously we found that Sema3a and its receptor PlxnA1 confer the high proliferative capacity and the resistance against epidermal growth factor receptor (EGFR) inhibitors to mouse-derived lewis lung cancer (LLC) cells constitutively expressing green fluorescent protein (GFP) (LLC-GFP). Here we show that PlxnA1 is essential for maintaining cancer stem-like property of LLC-GFP-derived cancer stem cells (LLC-GFPstem). Plxna1 knockdown downregulated stem cell markers, Sox2 and Cd44, in LLC-GFPstem cells. Importantly, the sphere-forming and tumorigenic capacity of LLC-GFPstem cells was lost by Plxna1 knockdown. These results demonstrate that PlxnA1 is essential for LLC-GFPstem cells to maintain their cancer stem-like properties.