Stenotrophomonas spp. intrinsically resistant to many β-lactam antibiotics are found throughout the environment. CESS-1 identified in Stenotrophomonas sp. KCTC 12332 is an uncharacterized class A β-lactamase. Here, CESS-1 was revealed to display hydrolytic activities toward penicillins (penicillin G and ampicillin) and cephalosporins (cephalexin, cefaclor, and cefotaxime), while its activity toward carbapenems (imipenem and meropenem) was negligible. Although cefaclor, cephalexin, and ampicillin have similar structures with identical R1 side chains, the catalytic parameters of CESS-1 toward the three β-lactam antibiotics were distinct. The k _cat values for cefaclor, cephalexin, and ampicillin were calculated to be 1249.6 s ^-1, 204.3 s ^-1, and 69.8 s ^-1, respectively, with the accompanying K _M values of 287.6 μM, 236.7 μM, and 28.8 μM, respectively. Remarkably, CESS-1 discriminates cefaclor and cephalexin with only one structural difference: -Cl (cefaclor) and -CH ₃ (cephalexin) at C3. According to structural comparisons among three E166Q mutants of CESS-1 acylated by cefaclor, cephalexin, and ampicillin, the cooperative positional changes of the R1 side chain of substrates and its contacting β5-β6 loop affect the distance between Asn170 and the deacylating water at the acyl-enzyme intermediate state. This is directly associated with the differential hydrolytic activities of CESS-1 toward the three structurally similar β-lactam antibiotics.