The impact of the antibiotic ampicillin (AMP) on the metabolic and kinetics of denitrification process as well as the sludge ability for oxidizing it was evaluated in batch assays. Denitrifying reference assays with acetate-C and nitrate-N (C/N ratio of 1.1) were conducted for establishing the metabolic and kinetic performance of the denitrifying sludge. Assays amended with 10 mg AMP-C L were also performed. In reference assays, acetate and nitrate consumption efficiencies of 100% with a total conversion to HCO and N were achieved within 1.5 h. When 10 mg AMP-C L was added, total and simultaneous consumption of nitrate-N, acetate-C, and AMP-C was achieved within 12 h. The substrates were completely reduced to N and oxidized to HCO, respectively. No nitrite-N was registered at the end of culture. AMP caused a reversible inhibitory effect on specific nitrate and acetate consumption and N production rates. Complete consumption and mineralization of AMP associated to nitrate reduction to N were achieved. This work provides the first evidences on the metabolic and kinetic performance of a denitrifying sludge exposed to AMP. These results might be considered for proposing useful wastewater treatments where β-Lactam antibiotics can be present.