Nano hydroxyapatite (nHAp) mimics the inorganic phase of hard tissue such as bone and teeth and, thus, has a wide range of clinical applications. The present study reports on the biomimetic synthesis of nHAp with and without Tris-buffered simulated body fluid (SBF) and investigated the role of buffering conditions on nHAp formation. The hypothesis of this study was that the nucleation and growth rate of nHAp may depend on buffering conditions during the precipitation process. The results of this study suggest that both of the above methods effectively synthesized carbonated "bone-like" nHAp. However, an increased incubation period of 8 hrs was necessary for nHAp synthesized using non Tris-buffered SBF as compared to Tris-buffered SBF which synthesized nHAp in just 3 hrs. Interestingly, there was no change in the chemical functionality for both samples. XRD and TGA analysis confirmed that Tris-buffered SBF facilitated more carbonate ion substitution than the non-Tris-buffered SBF approach. Therefore, this study concluded for the first time that the addition of Tris in SBF accelerates nHAp formation with more carbonate ion substitution. Nevertheless, carbonate ion substituted nHAp could also be synthesized using non Tris-buffered SBF, but would require longer incubation periods. This analysis highlights the importance of pH stability in the SBF for biomimetic nHAp synthesis which is useful for the synthesis of nHAp for a wide range of biomedical applications.