Conventional measurements of osteogenesis in tissue-engineered constructs are destructive to living cells and incapable to provide three-dimensional information. In the present study, noninvasive magnetic resonance (MR) microscopy was used to evaluate osteogenic differentiation in vitro in human mesenchymal stem cell-based tissue-engineered constructs. The constructs were prepared by seeding the cells (106cells/ml) on 4 × 4 × 4 mm gelatin sponge cubes and subsequently exposing them to osteogenic differentiation or basic medium. During the 4-week experiment, alkaline phosphatase (ALP) activity and calcium content of differentiated constructs were significantly increased compared to the basic medium controls. The T1, T2, and apparent diffusion coefficient (ADC) of differentiated constructs were significantly lower than those of the control group at each time point (p < 0.05). The MR parameters of constructs were significantly correlated to their ALP activities (r to T1, T2, and ADC = −0.57, −0.78, and −0.81, respectively) and calcium content (r to T1, T2, and ADC = 0.48, 0.90, and 0.92, respectively) measured by biochemical techniques. MR microscopy can be a promising tool for noninvasive assessment of osteogenic differentiation and to provide three-dimensional information about tissue-engineered constructs. © 2006 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res