AbstractCell separation is broadly useful for applications in clinical diagnostics, biological research, and potentially regenerative medicine. Recent attention has been paid to label‐free size‐based techniques that may avoid the costs or clogging issues associated with centrifugation and mechanical filtration. We present for the first time a massively parallel microfluidic device that passively separates pathogenic bacteria cells from diluted blood with macroscale performance. The device was designed to process large sample volumes in a high‐throughput, continuous manner using 40 single microchannels placed in a radial array with one inlet and two rings of outlets. Each single channel consists of a short focusing, gradual expansion and collection region and uses unique differential transit times due to size‐dependent inertial lift forces as a method of cell separation. The gradual channel expansion region is shown to manipulate cell equilibrium positions close to the microchannel walls, critical for higher efficiency collection. We demonstrate >80% removal of pathogenic bacteria from blood after two passes of the single channel system. The massively parallel device can process 240 mL/h with a throughput of 400 million cells/min. We expect that this parallelizable, robust, and label‐free approach would be useful for filtration of blood as well as for other cell separation and concentration applications from large volume samples. Biotechnol. Bioeng. 2010;107: 302–311. © 2010 Wiley Periodicals, Inc.