This study investigates the impact of short-duration spaceflight on human skin using multi-omics assays, including spatial transcriptomics, single-cell RNA/ATAC-seq, and metagenomics/metatranscriptomics. Skin biopsies were collected from the Inspiration4 crew members before and after a 3-day mission. The analysis revealed significant up-regulation of genes related to inflammation and KRAS signaling across all skin regions. These changes were associated with specific cellular responses, such as altered interferon responses, DNA damage, epithelial barrier disruptions, T-cell migration, and hindered regeneration, primarily in outer tissue compartments. The study also linked epithelial disruption to microbial shifts and immune cell activity in skin swabs and PBMC single-cell data. The findings provide insights into the molecular basis of skin changes during spaceflight and offer potential countermeasures for future space missions.This study investigates the impact of short-duration spaceflight on human skin using multi-omics assays, including spatial transcriptomics, single-cell RNA/ATAC-seq, and metagenomics/metatranscriptomics. Skin biopsies were collected from the Inspiration4 crew members before and after a 3-day mission. The analysis revealed significant up-regulation of genes related to inflammation and KRAS signaling across all skin regions. These changes were associated with specific cellular responses, such as altered interferon responses, DNA damage, epithelial barrier disruptions, T-cell migration, and hindered regeneration, primarily in outer tissue compartments. The study also linked epithelial disruption to microbial shifts and immune cell activity in skin swabs and PBMC single-cell data. The findings provide insights into the molecular basis of skin changes during spaceflight and offer potential countermeasures for future space missions.