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<i>Staphylococcus aureus</i> is an opportunistic human pathogen that is often involved in severe infections such as pneumonia and sepsis in which bacterial virulence factors play a key role. Infections caused by <i>S. aureus</i> are often difficult to eradicate, particularly when they are associated with biofilm. The physiological roles of the Crp/Fnr family regulator ArcR are elusive in <i>S. aureus</i>. In this study, it was found that the deletion of <i>arcR</i> increased the hemolytic ability and biofilm formation in <i>S. aureus</i>. Differential gene expression analysis by RNA-seq and real-time quantitative reverse transcription PCR showed that genes associated with hemolytic ability (<i>hla</i> and <i>hlb</i>) and biofilm formation (<i>icaA, icaB, icaC</i> and <i>icaD</i>) were significantly upregulated compared with those in the wild-type strain. The results revealed that ArcR regulated the expression of the <i>hla</i> and <i>ica</i> operon by binding to their promoter regions, respectively. This study provided new insights into the functional importance of ArcR in regulating the virulence and biofilm of <i>S. aureus.</i>