Outer space is an extreme environment and the survival of many microorganisms after spaceflight is well established. However, adaptations of Bacillus subtilis to space stress, particularly metabolism, are largely unknown. Here, we first performed a spaceflight mission of the B. subtilis TD7 strain and compared the spaceflight-exposed strain with the wild-type in terms of their phenotype, biofilm formation and secondary metabolism. The spaceflight-exposed strain exhibited slower growth, different morphology and decreased biofilm formation. Importantly, a decline in lipopeptide production was observed after spaceflight. Multi-omics approaches were used to uncover the molecular mechanisms underlying secondary metabolism and 997 differentially expressed genes (DEGs) were found, involving the TCA cycle, fatty acid degradation, amino acid biosynthesis and quorum sensing systems. Further analysis of 26 lipopeptide-related DEGs further elucidated the relationship between the space environment and secondary metabolism regulation. Our findings could contribute to a better understanding of the relationship between the space environment and microbial adaptation mechanisms.