Defective bone mineralization (osteomalacia) occurs in tissue non-specific alkaline phosphatase (TNAP) deficiency (hypophosphatasia). TNAP functions to maintain normal bone mineralization via hydrolysis of inorganic pyrophosphate (PPi), a bone mineralization inhibitor. This study sought to examine the role of PHOSPHO1, a soluble phosphatase that has specificity for phosphoethanolamine and phosphocholine, in increasing the inorganic phosphate (Pi)/inorganic pyrophosphate (PPi) ratio inside matrix vesicles (MVs) and, thus, controlling the first step of initiation of hydroxyapatite crystal deposition inside MVs. The identified molecular probe ML086 (CID-1674999) is a biochemical inhibitor of PHOSPHO1. PHOSPHO1-specific inhibitors may serve as novel tools to examine the functional role of this enzyme in skeletal mineralization and soft tissue ossification abnormalities, as well as to help elucidate its mechanism of action in diseases such as osteoarthritis, osteoporosis, and arterial calcification.