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Objectives We aimed to evaluate the effect of nanohydroxyapatite morphology and its interaction with anionic collagen on osteoblast activity. Materials and Methods Murine osteoblasts were incubated with a commercial collagen scaffold (as a control) or collagen-nanohydroxyapatite scaffolds (Col-HANP) for 24 and 48 hours for viability and proliferation assessments by MTT and Ki67 immunofluorescence, respectively. The hydroxyapatite nanoparticles were synthesized in three different morphologies/sizes (labeled as Col-HANP 0h, as Col-HANP 2h, and as Col-HANP 5h) as a function of the hydrothermal synthetic approach. Osteoblast's activity was investigated by bone alkaline phosphatase activity (ALP) and Von Kossa mineralization assays. For biocompatibility evaluation, the scaffolds were implanted subcutaneously in the dorsum of male Wistar rats for 7 and 15 days. Results The incubation of cells with Col-HANP 5h for 48h resulted in a significant increase in their proliferation and activity. The implantation of Col-HANP 5h in the subcutaneous tissue presented decreased recruitment of inflammatory cells and IL-1β levels on day 7, as well as an increase in collagen synthesis on day 15 compared to collagen and control groups. Conclusions The significant effects on osteoblasts proliferation and activity illustrate the potential application of Col-HANP 5h scaffold as a promising strategy for bone tissue engineering.