In this study, polypyrrole (Ppy) electrodes were prepared to support an electrical stimulation to MC3T3-E1 cells for regulating their osteogenic differentiation. The charge injection capacity (C_Q) of the Ppy electrodes could be adjusted by the Ppy thickness, and a higher C_Q could make the electrode able to produce a higher charge injection quantity (Q_inj) at applied voltage. The Q_inj onto electrode could be considered as the intensity of the stimulation pulse to cells, and the pulse frequency means the number of electric stimulation with Q_inj at one second. Hence, we conducted the present work in the view of Q_inj. When the cells were electrically stimulated for 1 hour per day, the electrodes with Q_inj ranged in 0.08–0.15 μQ had an obvious role in enhancing cellular osteogenic differentiation whereas Q_inj of lower than 0.03 μQ or more than 0.30 μQ gave the stimulations with no or negative effects. And the stimulation with 1 or 25 Hz showed to enhance the differentiation, whereas the stimulation with 50 Hz gave an inhibiting effect. We further found the osteogenic differentiation potential triggered by electrical simulation was related to cell growth stage, and the stimulation carried out at early stage (day 2–5) during 8 days cell culture showed more contribution to enhancing osteogenic differentiation than that at later stage (day 6–8). It is proposed that the desired stimulation effects require that an appropriate voltage-gated calcium ion channel and efficient intracellular calcium ion oscillation are well activated. This work therefore reveals Q_inj as an important electrode parameter to decide effective simulations and provides an insight into understanding of the role of electrode material characters in regulating cellular osteogenic differentiation during stimulation.