Infections caused by the leading nosocomial pathogen Staphylococcus epidermidis are characterized by biofilm formation on implanted medical devices. However, the molecular basis of biofilm formation and its regulation are not completely understood. Here, we describe an important role of the ClpP protease in biofilm development and virulence of S. epidermidis. We constructed an isogenic clpP mutant strain of a biofilm-forming clinical isolate of S. epidermidis. The mutant strain showed decreased biofilm formation in vitro and reduced virulence in a rat model of biofilm-associated infection. Biofilm forming ability of the mutant strain could be restored by expressing clpP on a plasmid, but not when a catalytically inactive allele of clpP gene was introduced. These observations indicate that the peptidase function of ClpP determines its role in biofilm formation. Experimental data in this work also suggested that clpP influenced initial attachment of bacteria on the plastic surface, the first step of biofilm formation. Furthermore, clpP was found to be regulated by the quorum-sensing agr, suggesting that part of the previously described influence of agr on the initial attachment to plastic surfaces may be mediated by clpP.