Synthetic hydroxyapatite nanoparticles (nHAp) possess compositional and structural similarities to those of bone minerals and play a key role in bone regenerative medicine. Functionalization of calcium phosphate biomaterials with Sr, i.e., bone extracellular matrix trace element, has been proven to be an effective biomaterial-based strategy for promoting osteogenesis in vitro and in vivo. Functionalizing nHAp with Sr2+ ions or strontium ranelate (SrRAN) can provide favorable bone tissue regeneration by locally delivering bioactive molecules to the bone defect microenvironment. Moreover, administering an antiosteoporotic drug, SrRAN, directly into site-specific bone defects could significantly reduce the necessary drug dosage and the risk of possible side effects. Our study evaluated the impact of the Sr source (Sr2+ ions and SrRAN) used to functionalize nHAp by wet precipitation on its in vitro cellular activities. The systematic comparison of physicochemical properties, in vitro Sr2+ and Ca2+ ion release, and their effect on in vitro cellular activities of the developed Sr-functionalized nHAp was performed. The ion release tests in TRIS-HCl demonstrated a 21-day slow and continuous release of the Sr2+ and Ca2+ ions from both Sr-substituted nHAp and SrRAN-loaded HAp. Also, SrRAN and Sr2+ ion release kinetics were evaluated in DMEM to understand their correlation with in vitro cellular effects in the same time frame. Relatively low concentration (up to 2 wt %) of Sr in the nHAp led to an increase in the alkaline phosphatase activity in preosteoblasts and expression of collagen I and osteocalcin in osteoblasts, demonstrating their ability to boost bone formation.