Calcific tendonitis is a frequent cause of chronic shoulder pain. Its cause is currently poorly known. The objectives of this study were to better characterize the cells and mechanisms involved in depositing apatite crystals in human tendons. Histologic sections of cadaveric calcified tendons were analyzed, and human calcific deposits from patients undergoing lavage of their calcification were obtained to perform infrared spectroscopy and mass spectrometry-based proteomic characterizations. In vitro, the mineralization ability of human rotator cuff cells from osteoarthritis donors was assessed by alizarin red or Von Kossa staining. Calcifications were amorphous areas surrounded by a fibrocartilaginous metaplasia containing hypertrophic chondrocyte-like cells that expressed tissue non-specific alkaline phosphatase (TNAP) and ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), which are two key enzymes of the mineralization process. Calcific deposits were composed of apatite crystals associated with proteins involved in bone and cartilage development and endochondral bone growth. In vitro, tenocyte-like cells extracted from the rotator cuff were able to mineralize in osteogenic cultures, and expressed TNAP, type X COLLAGEN, and MMP13, which are hypertrophic chondrocytes markers. The use of a TNAP inhibitor significantly prevented mineral deposits. We provide evidence that tenocytes have a propensity to differentiate into hypertrophic chondrocyte-like cells to produce TNAP-dependent calcium deposits. We believe that these results may pave the way to identifying regulating factors that might represent valuable targets in calcific tendonitis