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Human tongue epithelium is one of the most proliferative epithelia and one of the most common sites for oral cancers, suggesting its robust stem cell activity. However, the cellular and molecular heterogeneity within human tongue epithelium is still elusive. We performed single-cell RNA sequencing to characterize cellular and molecular heterogeneity within human tongue epithelium. By analyzing differentially expressed genes and genetic signatures in the basal, proliferative, and suprabasal cells, we selected potential cell surface markers for human lingual stem cells and differentiated cells. We further show that the TGF-β signaling pathway is differentially activated between basal and suprabasal cells and regulates human lingual stem cell self-renewal and differentiation. This comprehensive atlas of human lingual epithelium provides an important reference for the study of lingual stem cells and regenerative medicine.