A better understanding of the biology of tissue-resident stem cell populations is essential to development of therapeutic strategies for regeneration of damaged tissue. Here, we describe the isolation of glandular stem cells (GSCs) from a small biopsy specimen from human parotid glands. Single colony-forming unit-derived clonal cells were isolated through a modified subfractionation culture method, and their stem cell properties were examined. The isolated clonal cells exhibited both epithelial and mesenchymal stem cell (MSC)-like features, including differentiation potential and marker expression. The cells transiently displayed salivary progenitor phenotypes during salivary epithelial differentiation, suggesting that they may be putative multipotent GSCs rather than progenitor cells. Both epithelial and mesenchymal-expressing putative GSCs, LGR5+CD90+ cells, were found in vivo, mostly in inter-secretory units of human salivary glands. Following in vivo transplantation into irradiated salivary glands of mice, these cells were found to be engrafted around the secretory complexes, where they contributed to restoration of radiation-induced salivary hypofunction. These results showed that multipotent epitheliomesenchymal GSCs are present in glandular mesenchyme, and that isolation of homogenous GSC clones from human salivary glands may promote the precise understanding of biological function of bona fide GSCs, enabling their therapeutic application for salivary gland regeneration.