Better quantitative understanding of human mesenchymal stem cells (hMSCs) metabolism is needed to identify, understand, and subsequently optimize the processes in expansion of hMSCs in vitro. For this purpose, we analyzed growth of hMSCs in vitro with a mathematical model based on the mass balances for viable cell numbers, glucose, lactate, glutamine, and glutamate. The mathematical modeling had two aims: (1) to estimate kinetic parameters of important metabolites for hMSC monolayer cultures, and (2) to quantitatively assess assumptions on growth of hMSCs. Two cell seeding densities were used to investigate growth and metabolism kinetics of MSCs from three human donors. We analyzed growth up to confluency and used metabolic assumptions described in literature. Results showed a longer initial phase, a slower growth rate, and a higher glucose, lactate, glutamine, and glutamate metabolic rates at the lower cell seeding density. Higher metabolic rates could be induced by a lower contact inhibition effect when seeding at 100cells/cm2 than when seeding at 1000cells/cm2. In addition, parameter estimation describing kinetics of hMSCs in culture, depending on the seeding density, showed doubling times in the order of 17–32h, specific glucose consumption in the order of 1.25×10−1 to 3.77×10−1pmol/cell/h, specific lactate production in the order of 2.48×10−1 to 7.67×10−1pmol/cell/h, specific glutamine production in the order of 7.04×10−3 to 2.27pmol/cell/h, and specific glutamate production in the order of 4.87×10−1 to 23.4pmol/cell/h. Lactate-to-glucose yield ratios confirmed that hMSCs use glucose via anaerobic glycolysis. In addition, glutamine and glutamate metabolic shifts were identified that could be important for understanding growth of hMSCs in vitro. This study showed that the mathematical modeling approach supports quantitative analysis of important mechanisms in proliferation of hMSCs in vitro.