Sweat glands perform a vital thermoregulatory function in mammals. Like other skin components, they originate from epidermal progenitors. However, they have low regenerative potential in response to injury. We have established a sweat gland culture and expansion method using 3D organoids cultures. The epithelial cells derived from sweat glands in dermis of adult mouse paw pads were embedded into Matrigel and formed sweat gland organoids (SGOs). These organoids maintained remarkable stem cell features and demonstrated differentiation capacity to give rise to either sweat gland cells (SGCs) or epidermal cells. Moreover, the bipotent SGO-derived cells could be induced into stratified epidermis structures at the air-liquid interface culture in a medium tailored for skin epidermal cells in vitro. The SGCs embedded in Matrigel tailored for sweat glands formed epithelial organoids, which expressed sweat-gland-specific markers, such as cytokeratin (CK) 18 and CK19, aquaporin (AQP) 5 and αATP. More importantly, they had potential of regeneration of epidermis and sweat gland when they were transplanted into the mouse back wound and claw pad with sweat gland injury, respectively. In summary, we established and optimized culture conditions for effective generation of mouse SGOs. These cells are candidates to restore impaired sweat gland tissue as well as to improve cutaneous skin regeneration.