The objective of the RR-23 mission was to better understand the effects of spaceflight on the eyes, specifically on the structure and function of the arteries, veins, and lymphatic vessels that are needed to maintain vision. To this end, twenty male, C57BL/6J, 16-17 weeks-old mice were delivered to the ISS on SpaceX-21 in a single transporter, transferred to two rodent habitats, and maintained in microgravity for 38 days. Flight mice were then returned to Earth alive (Jan 13th, 2021). After splashdown in the Atlantic Ocean, mice were transported to Kennedy Space Center via helicopter. The 20 Flight, 20 Habitat Ground Control (HGC), and 20 Vivarium Ground Control (VGC) mice were removed from Rodent Transporters (Flight and HGC) or vivarium cages (VGC), placed into shipping containers, and flown to Texas A and M University. There, mice underwent post-flight procedures, before euthanasia and tissue collection. Flight, HGC and VGC animals were euthanized and dissected on Jan 14th, 17th or 20th of 2021, respectively. Feces were removed from the animals and preserved by flash freezing in liquid nitrogen and stored at -80 ˚C until DNA was extracted, and libraries generated and sequenced (target 10 M clusters per sample, PE 250 bp). This dataset features 9 samples from the Flight group, 9 samples from the HGC group, and 9 samples from the VGC group.