The role of microvesicles (MVs) in transcellular signal transduction has been demonstrated in different studies. However, the potential modulatory role of MVs in fracture healing remains unclear. Therefore, we investigated the impact of plasma-derived MVs after a femoral fracture on cranial osteoblasts. A femoral fracture with intramedullary stabilization was induced in Sprague Dawley rats. The animals were killed 3 days (group A), 1 week (group B), or 2 weeks (group C) after trauma induction. Animals without trauma served as controls. Osteoblasts from the cranial bone of a neonatal Sprague Dawley rats were cultured and stimulated with either plasma-derived MVs or MV-free plasma of groups A to C. The effects of MVs on osteoblasts were analyzed by growth assay, metabolic assay, and quantitative real-time polymerase chain reaction for osteocalcin, RUNX2, and collagen 1A to test differentiation of osteoblasts. MVs were time-dependently incorporated in osteoblasts and localized mainly around the nucleus. MVs increased the viability of osteoblasts, particularly in the late phase after femoral fracture (group A, P = 0.0276; group B, P = 0.0295; group C, P = 0.0407). Late-phase differentiation of osteoblasts was not stimulated by MVs but was by MV-free plasma (osteocalcin, groups C vs. control, P = 0.0454). The levels of transforming growth factor β1 (P = 0.0320) and insulin-like growth factor 1 ( P = 0.0211) were significantly higher in plasma than in MVs. MVs seem to modulate the viability of osteoblasts but not to affect osteoblast differentiation. Further studies are warranted to determine the characteristics and interactions of MVs. Potentially, MVs might act as a diagnostic or therapeutic tool in cases of impairment of fracture healing.