In our study, Bacillus subtilis was disposed to a simulated microgravity (SMG) environment in high-aspect ratio rotating-wall vessel bioreactors for 14 days, while the control group was disposed to the same bioreactors in a normal gravity (NG) environment for 14 days. The B. subtilis strain exposed to the SMG (labeled BSS) showed an enhanced growth ability, increased biofilm formation ability, increased sensitivity to ampicillin sulbactam and cefotaxime, and some metabolic alterations compared with the B. subtilis strain under NG conditions (labeled BSN) and the original strain of B. subtilis (labeled BSO). The DEPs associated with an increased growth rate, such as DNA strand exchange activity, oxidoreductase activity, proton-transporting ATP synthase complex, and biosynthetic process, were significantly upregulated in BSS. The enhanced biofilm formation ability may be related with the DEPs of spore germination and protein processing in BSS, and DEGs involved in protein localization, peptide secretion were also significantly enriched. The results revealed that SMG may increase the level of related functional proteins by upregulating or downregulating affiliated genes to change physiological characteristics and modulate growth ability, biofilm formation ability (epsB, epsC, epsN), antibiotic sensitivity (penP) and metabolism. Our experiment may gives new ideas for the study of space microbiology.