Effective procedures for the purification of Leydig cells (LCs) can facilitate functional studies and transplantation therapies. However, current methods to purify LCs from testes are still far from satisfactory. Here, we found that testicular autofluorescence existed in the interstitium along with the gradual maturation of LCs from birth to adulthood. These autofluorescent cells were further isolated by fluorescence-activated cell sorting (FACS) and determined to be composed of LCs and macrophages. To further purify LCs, we combined two fluorescence channels of FACS and successfully separated LCs and macrophages. Of note, we confirmed that the obtained LCs not only possessed high purity, viability and quantity but also had intact steroidogenic activity and excellent responsiveness to luteinizing hormone. Moreover, subcutaneous transplantation of isolated LCs could alleviate the symptoms of testosterone deficiency in castrated mice. In summary, we established an effective autofluorescence-based method for isolating LCs. This method will aid in the future success of using LCs for basic and translational applications. • Testicular autofluorescent cells composed of macrophages and LCs. • The autofluorescence-based method by combining two fluorescence channels in FACS could separate LCs from macrophages. • The isolated LCs had high purity, viability and quantity. • The isolated LCs retained the intact steroidogenic activity and exhibited an outstanding responsiveness to LH. • Subcutaneous transplantation of the isolated LCs could alleviate the symptoms of TD in castrated mice. [ABSTRACT FROM AUTHOR]