Depletion of microbiota increases susceptibility to gastrointestinal colonization and subsequent infection by opportunistic pathogens such as methicillin-resistant Staphylococcus aureus (MRSA). How the absence of gut microbiota impacts the evolution of MRSA is unknown. The present report used germ-free mice to investigate the evolutionary dynamics of MRSA in the absence of gut microbiota. Through genomic analyses and competition assays, we found that MRSA adapts to the microbiota-free gut through sequential genetic mutations and structural changes that enhance fitness. Initially, these adaptations increase carbohydrate transport; subsequently, evolutionary pathways largely diverge to enhance either arginine metabolism or cell wall biosynthesis. Increased fitness in arginine pathway mutants depended on arginine catabolic genes, especially nos and arcC, which promote microaerobic respiration and ATP generation, respectively. Thus, arginine adaptation likely improves redox balance and energy production in the oxygen-limited gut environment. Findings were supported by human gut metagenomic analyses, which suggest the influence of arginine metabolism on colonization. Surprisingly, these adaptive genetic changes often reduced MRSA's antimicrobial resistance and virulence. Furthermore, resistance mutation, typically associated with decreased virulence, also reduced colonization fitness, indicating evolutionary trade-offs among these traits. The presence of normal microbiota inhibited these adaptations, preserving MRSA's wild-type characteristics that effectively balance virulence, resistance, and colonization fitness. The results highlight the protective role of gut microbiota in preserving a balance of key MRSA traits for long-term ecological success in commensal populations, underscoring the potential consequences on MRSA's survival and fitness during and after host hospitalization and antimicrobial treatment.