The separation of motile sperm from semen samples is sought after for medical infertility treatments. In this work, we demonstrate a high-throughput microfluidic device that can passively isolate motile sperm within corrals inside a fluid channel, separating them from the rest of the diluted sample. Using finite element method simulations and proposing a model for sperm motion, we investigated how flow rate can provide a rheotaxis zone in front of the corral for sperm to move upstream/downstream depending on their motility. Using three different flow rates that provided shear rates above the minimum value within the rheotaxis zone, we experimentally tested the device with human and bovine semen. By taking advantage of the rheotactic behavior of sperm, this microfluidic device is able to corral motile sperm with progressive velocities in the range of 48–93 μm⋅s −1 and 51–82 μm⋅s −1 for bovine and human samples, respectively. More importantly, we demonstrate that the separated fractions of both human and bovine samples feature 100% normal progressive motility. Furthermore, by extracting the sperm swimming distribution within the rheotaxis zone and sperm velocity distribution inside the corral, we show that the minimum velocity of the corralled sperm can be adjusted by changing the flow rate; that is, we are able to control the motility of the separated sample. This microfluidic device is simple to use, is robust, and has a high throughput compared with traditional methods of motile sperm separation, fulfilling the needs for sperm sample preparation for medical treatments, clinical applications, and fundamental studies.