Most clinically relevant methicillin-resistant Staphylococcus aureus (MRSA) strains have become resistant to β-lactams antibiotics through horizontal acquisition of the mecA gene encoding PBP2a, a peptidoglycan transpeptidase with low affinity for β-lactams. The level of resistance conferred by mecA is, however, strain dependent, and the mechanisms underlying this phenomenon remain poorly understood. We show here that β-lactam resistance correlates to expression of the Sle1 cell wall amidase in the fast-spreading and highly virulent community-acquired MRSA USA300 clone. Sle1 is a substrate of the ClpXP protease, and while the high Sle1 levels in cells lacking ClpXP activity confer β-lactam hyper-resistance, USA300 cells lacking Sle1 are as susceptible to β-lactams as cells lacking mecA. This finding prompted us to assess the cellular roles of Sle1 in more detail, and we demonstrate that high Sle1 levels accelerate the onset of daughter cells splitting and decrease cell size. Vice versa, oxacillin decreases the Sle1 level and imposes a cell separation defect that is antagonized by high Sle1 levels, suggesting that high Sle1 levels increase tolerance to oxacillin by promoting cell separation. In contrast, increased oxacillin sensitivity of sle1 cells appears linked to a synthetic lethal effect on septum synthesis. In conclusion, this study demonstrates that Sle1 is a key factor in resistance to β-lactam antibiotics in the JE2 USA300 model strain and that PBP2a is required for the expression of Sle1 in JE2 cells exposed to oxacillin.