Aldehyde dehydrogenase 1 (ALDH1) activity has now been employed successfully as a cancer stem cells (CSCs) marker in various tumors. We wanted to investigate whether ALDH1 can be used as a putative CSCs marker and a powerful prognostic factor in nasopharyngeal carcinoma (NPC). Here, we isolated ALDH1-positive cells from human NPC cell lines (5-8F and CNE2) and found that ALDH1-positive cancer cells grew faster and had higher clone formation efficiency (0.435±0.15; 0.431±0.025 vs. 0.131±0.007; 0.121±0.126), differentiation capability and had higher migration (233.00±5.29; 228.60±9.34 vs. 123.20±7.70 vs. 97.20±6.61) than those of ALDH1-negative cancer cells in vitro. In addition, ALDH1- positive cancer cells formed significantly more tumor spheres. Our in vivo experiments showed that only 5×10(3) ALDH1-positive NPC cells were required to induce tumors. Notably, ALDH1-positive cells are enriched in the side-population (SP) cells, and stem cells-like genes OCT4, BMI1, KLF4 and SOX2 are preferentially expressed in ALDH1-positive cells. Immunohistochemical results showed that the expression of ALDH1 correlated significantly with T classification (P=0.011), N classification (P=0.005), M classification (P=0.002) and clinical stage (P=0.001). Interestingly, ALDH1 expression in the tumor correlated significantly with epithelial-mesenchymal transition (EMT) markers including vimentin expression and loss of E-cadherin (P=0.003 and 0.008, respectively). Furthermore, multivariate analyses showed that ALDH1 expression was an independent prognostic indicator (P=0.032). Taken together, for the first time, we demonstrate that ALDH1 could be a novel stem cells marker and a valuable predictor of poor survival NPC.