Abstract Acidosis characterizes the microenvironment of most solid tumors and is considered a new hallmark of cancer. It is mainly caused by both “aerobic” and “anaerobic” glycolysis of differently adapted cancer cells, with the final product lactic acid being responsible of the extracellular acidification. Many evidences underline the role of extracellular acidosis in tumor progression. Among the different findings, we demonstrated that acidosis-exposed cancer cells are characterized by an epithelial-to-mesenchymal transition phenotype with high invasive ability, high resistance to apoptosis, anchorage-independent growth, and drug therapy. Acidic melanoma cells over-express SOX2, which is crucial for the maintenance of their oxidative metabolism, and carbonic anhydrase IX, that correlates with poor prognosis of cancer patients. Considering these evidences, we realized that the profile outlined for acid cancer cells inevitably remind us the stemness profile. Therefore, we wondered whether extracellular acidosis might induce in cancer cells the acquisition of stem-like properties and contribute to the expansion of the cancer stem cell sub-population. We found that a chronic adaptation to acidosis stimulates in cancer cells the expression of stem-related markers, also providing a high in vitro/in vivo clonogenic and trans-differentiating ability. Moreover, we observed that the acidosis-induced stem-like phenotype of melanoma cells was reversible and related to the EMT induction. These findings help to characterize a further aspect of stem cell niche, contributing to the sustainment and expansion of cancer stem cell subpopulation. Thus, the usage of agents controlling tumor extracellular acidosis might acquire great importance in the clinic for the treatment of aggressive solid tumor. Key messages • Extracellular acidosis up-regulates EMT and stem-related markers in melanoma cells • Acidic medium up-regulates in vitro self-renewal capacity of melanoma cells • Chronic acidosis adaptation induces trans-differentiation ability in melanoma cells • Melanoma cells adapted to acidosis show higher tumor-initiating potential than control cells • Extracellular acidosis promotes a stem-like phenotype in prostate and colorectal carcinoma cells