In Rodent Research Reference Mission-1 (RRRM-1), forty female BALB/cAnNTac mice were flown on the International Space Station. To assess differences in outcomes due to age, twenty 10-12 week-old and twenty 32 week-old mice were flown, respectively. To directly assess spaceflight effects, half of the young and old mice (10 old, 10 young) were sacrificed on-orbit after 22-23 days (ISS Terminal, ISS-T), while the other half (10 old, 10 young) were returned live to Earth after 40 days and allowed to recover for 2 days (Live Animal Return, LAR) before sacrifice. Both the ISS-T and LAR animals had independent ground controls (10 mice housed in flight hardware in matched environmental conditions), basal controls (10 mice sacrificed 1 day after launch), and vivarium controls (10 mice housed within standard vivarium habitats). Thus RRRM-1 included a total of 160 mice. Feces were removed from 145 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 251 bp). This dataset features whole-genome shotgun metagenomics sequencing data from 8-10 feces samples per group.