Microfluidic trapping methods create significant opportunities to establish highly controlled cell positioning and arrangement for the microscale study of numerous cellular physiological and pathological activities. However, a simple, straightforward, dynamic, and high-throughput method for cell trapping is not yet well established. In the present paper, we report a direct active trapping method using an integrated microfluidic device with pneumatic microstructures (PμSs) for both operationally and quantitatively dynamic localization of cells, as well as for high-throughput cell patterning. We designed and fabricated U-shape PμS arrays to replace the conventional fixed microstructures for reversible trapping. Multidimensional dynamics and spatial consistency of the PμSs were optically characterized and quantitatively demonstrated. Furthermore, we performed a systematic trapping investigation of the PμSs actuated at a pressure range of 0 psi to 20 psi using three types of popularly applied mammalian cells, namely, human lung adenocarcinoma A549 cells, human hepatocellular liver carcinoma HepG2 cells, and human breast adenocarcinoma MCF-7 cells. The cells were quantitatively trapped and controlled by the U-shape PμSs in a programmatic and parallel manner, and could be opportunely released. The trapped cells with high viability were hydrodynamically protected by the real-time actuation of specifically designed umbrella-like PμSs. We demonstrate that PμSs can be applied as an active microfluidic component for large-scale cell patterning and manipulation, which could be useful in many cell-based tissue organization, immunosensor, and high-throughput imaging and screening.