In the field of bone tissue engineering, there is a need for materials that mimic the native bone extracellular matrix (ECM). This need is met through the creation of biphasic composites intended to mimic both the organic and inorganic facets of the native bone ECM. However, few studies have created composites with organic ECM analogous components capable of directing cellular behaviors and many are not fabricated in the nanoscale. Furthermore, few attempts have been made at investigating how variations of organic and inorganic components affect the osteogenic differentiation of human mesenchymal stem cells (hMSCs). To address these issues, biphasic nanomatrix composites consisting of hydroxyapatite nanoparticles (HANPs) embedded within peptide amphiphile (PA) nanofibers tailored with the RGDS cellular adhesion motif (PA-RGDS) were created at various HANP to PA-RGDS ratios. Fabrication of these biphasic nanomatrix composites was confirmed via scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The long-term cellularity and osteogenic differentiation of hMSCs in response to the different compositional ratios were then assessed by quantifying the timed expression of genes indicative of osteogenic differentiation, alkaline phosphatase activity, and DNA content over time. Decreased cellularity and the expression of genes over time correlated with increasing compositional ratios between HANP and PA-RGDS. The highest HANP to PA-RGDS ratio (66% HANP) exhibited the greatest improvement to the osteogenic differentiation of hMSCs. Overall, these results demonstrate that the compositional ratio of biphasic nanomatrix composites plays an important role in influencing the osteogenic differentiation of hMSCs. Based on the observations presented within this study, these biphasic nanomatrix composites show promise for future usage in bone tissue engineering applications. © 2012 Acta Materialia Inc.