The Clp protease complex degrades a multitude of substrates, which are engaged by a AAA+ chaperone such as ​ClpX and subsequently digested by the dynamic, barrel-shaped ​ClpP protease. Acyldepsipeptides (ADEPs) are natural product-derived antibiotics that activate ​ClpP for chaperone-independent protein digestion. Here we show that both protein and small-molecule activators of ​ClpP allosterically control the ​ClpP barrel conformation. We dissect the catalytic mechanism with chemical probes and show that ADEP in addition to opening the axial pore directly stimulates ​ClpP activity through cooperative binding. ​ClpP activation thus reaches beyond active site accessibility and also involves conformational control of the catalytic residues. Moreover, we demonstrate that substoichiometric amounts of ADEP potently prevent binding of ​ClpX to ​ClpP and, at the same time, partially inhibit ​ClpP through conformational perturbance. Collectively, our results establish the hydrophobic binding pocket as a major conformational regulatory site with implications for both ClpXP proteolysis and ADEP-based anti-bacterial activity.