Aim: This study aimed to determine the effect of mechanical ventilation (MV) on the differentiation and proliferation of diaphragm satellite cells. Methods: Diaphragm satellite cells were isolated from C57 mice receiving 6 h of MV with optimized magnetic-activated cell sorting (MACS) approach. The cells were stained with BrdU or antibody for differentiation marker MYH3. The expression of MyoD and myogenin was detected by real-time PCR. Results: Diaphragm satellite cells were successfully isolated from mice by using MACS with a set of optimized parameters. About 1.5 \times 10{}^5 cells could be harvested from a diaphragm. Upon MV, the proliferation rate of diaphragm satellite cells was decreased from 88.74% to 81.92%, while the differentiation rate was increased from 17.94% to 27.58%, compared to controls. Moreover, the expression of MyoD and myogenin were significantly upregulated upon MV. Conclusions: We established a practical method to purify diaphragm satellite cells, and demonstrated that MV regulated the differentiation and proliferation of diaphragm satellite cells.