High sperm DNA damage is associated with decreased normal fertilization, embryo development and pregnancy rates, and an increased miscarriage rate. Thus, selecting sperm with minimal DNA damage is important for assisted reproductive technology. A microfluidic sperm selection chamber (MSS, ZyMōt™; DxNow) is a device designed to collect sperm with high motility and chromatin integrity, which has recently been clinically used for ICSI/IVF. Here we aimed to comprehensively evaluate MSS compared to density-gradient centrifugation (DGC) in terms of (1) sperm DNA fragmentation by sperm chromatin dispersion (SCD) test and comet assay, (2) sperm motility and morphological features of motile sperm, and (3) blastocyst development after ICSI and clinical outcomes after vitrified-thawed single blastocyst transfers (vSBT). Sperm analysis was performed in 23 cases between October 2020 and February 2021. SCD test was optimized as a rapid procedure (≤30 min), with sperm showing a halo deemed normal, and those without a halo abnormal. Comet assay results were analyzed using CometScore 2.0, with comparison of %Tail DNA, Tail Length (TL), and Tail Moment (TM). Sperm motility parameters were investigated using the Sperm Motility Analysis System (SMAS, Dietect). To assess motile spermatozoa characteristics, middle piece morphology and active mitochondrial distribution were labeled by MitoTracker-Red. The reactive oxygen species (ROS) level was labeled using CM-H2DCFDA. ICSI outcomes (normal fertilization, blastocyst development, and pregnancy rates) were compared between DGC and MSS for 620 cycles between September 2019 and December 2020. In SCD test, MSS yielded a significantly lower abnormality rate than for raw sperm and DGC in all cases. The comet assay showed that %Tail DNA, TL, and TM were significantly lower with MSS than for raw sperm or DGC. Sperm motility rates were significantly higher with DGC and MSS than for raw sperm. Sperm motility analysis demonstrated significantly lower straightness, linearity, and beat-cross frequency (Hz) with MSS compared to DGC. The morphological abnormality rate of the sperm midpiece was significantly higher with MSS than DGC. Abnormal sperm midpieces exhibited significantly high ROS activity around mitochondria. Fertilization after ICSI and blastocyst rates were similar between MSS and DGC, while the vitrified blastocyst rate was significantly higher with MSS than DGC. Clinical pregnancy and miscarriage rates after vSBT did not significantly different between MSS and DGC. MSS-selected sperm had high DNA integrity and were homogeneous. The sperm had a higher motility rate, but the characteristic sperm motility pattern was meandering motion. Additionally, MSS did not exclude sperm that were morphologically abnormal in the middle piece with high ROS levels. MSS-ICSI yielded an improved vitrified blastocyst rate, and MSS is an effective device for sperm collection for ICSI.