In this paper, it has been studied the acclimation stage of a synthetic wastewater fed with glucose as a carbon source, using a tow-chambers microbial fuel cells (MFCs). Special attention has been paid to the start-up. During the acclimation period, the microbial fuel cells (MFCs) will be exposed to variations in operating parameters. Hence, the acclimation stage of MFCs, exposed to variation in the influent COD, operating temperature, and electron acceptor, was investigated in the terms of power density, COD removal efficiency, and voltage while treating a synthetic wastewater. The power density is increased and the acclimation period is prolonged with the increase of the influent COD up to meet steady-state conditions. It is important to note that the acclimation of MFCs is not only impacted by the electricity-generating bacteria, but by the whole biological. The highest steady-state voltage, which is about 404mV, is obtained at 35°C, comparing to the operating temperature of 15°C or 25°C. In addition, the electron acceptor will obviously influence the steady-state voltage and start-up period.