The multi-substituted hydroxyapatites (ms-HAPs) are currently gaining more consideration due to their multifunctional properties and biomimetic structure, owning thus an enhanced biological potential in orthopedic and dental applications. In this study, nano hydroxyapatite (HAP) substituted with multiple cations (Sr2+, Mg2+ and Zn2+) for Ca2+ and anion (SiO44-) for PO43- and OH-, specifically HAPc-5%Sr and HAPc-10%Sr (where HAPc is HAP-1.5%Mg-0.2%Zn-0.2%Si), both lyophilized noncalcined and lyophilized calcined, were evaluated for their in vitro ions release. These nanomaterials were characterized by SEM, FE-SEM and EDX, as well as by AFM images and by surface-specific areas and porosity. Further, the release of cations and of phosphate anions were assessed from nano HAP and ms-HAPs, both in water and in simulated body fluid (SBF), in static and simulated dynamic conditions, using inductively coupled plasma optical emission spectrometry (ICP-OES). The release profiles were analyzed and the influence of experimental conditions was determined for each of the six nanomaterials and for various periods of time. The pH of the samples soaked in the immersion liquids was also measured. The ions release mechanism was theoretically investigated using the Korsmeyer-Peppas model. The results indicated a mechanism principally based on diffusion and dissolution, with possible contribution of ion exchange. The surface of ms-HAP nanoparticles is more susceptible to dissolution into immersion liquids due to the lattice strain provoked by simultaneous multi-substitution in HAP structure. According to the findings, it is rational to suggest that both materials HAPc-5%Sr and HAPc-10%Sr are bioactive and can be potential candidates in bone tissue regeneration.