Human mesenchymal stem cells (hMSCs) possess potential of bone formation and were proposed as ideal material against osteoporosis. Although interrogation of directing effect on lineage specification by physical cues has been proposed, how mechanical stimulation impacts intracellular viscoelasticity during osteogenesis remained enigmatic. Cyto-friendly 3D matrix was prepared with polyacrylamide and conjugated fibronectin. The hMSCs were injected with fluorescent beads and chemically-induced toward osteogenesis. The mechanical properties were assessed using video particle tracking microrheology. Inverted epifluorescence microscope was exploited to capture the Brownian trajectory of hMSCs. Mean square displacement was calculated and transformed into intracellular viscoelasticity. Two different stiffness of microspheres (12 kPa, 1 kPa) were established. A total of 45 cells were assessed. hMSCs possessed equivalent mechanical traits initially in the first week, while cells cultured in rigid matrix displayed significant elevation over elastic (G’) and viscous moduli (G”) on day 7 (p p < 0.01). However, after two weeks, soft niches no longer stiffened hMSCs, whereas the effect by rigid substrates was consistently during the entire differentiation course. Stiffness of matrix impacted the viscoelasticity of hMSCs. Detailed recognition of how microenvironment impacts mechanical properties and differentiation of hMSCs will facilitate the advancement of tissue engineering and regenerative medicine.