OBJECTIVE:Antimicrobial resistance in pathogens causing bovine respiratory disease (BRD) is promoted by the prophylactic use of antimicrobials in calves. However, the impact of discontinuing their use under field conditions remains unclear. This study evaluated changes in antimicrobial susceptibility of Mannheimia haemolytica and Pasteurella multocida following cessation of prophylactic oxytetracycline and tilmicosin on a contract-rearing farm for Holstein heifer calves in Japan. METHODS:Oxytetracycline was administered via milk replacer at 20 mg/kg/d to calves aged 6-60 d of age and discontinued in April 2022, whereas prophylactic tilmicosin (10 mg/kg, single subcutaneous injection at arrival) was discontinued in May 2023. From November 2021 to November 2023, 850 nasal and ear swab samples were collected from 583 calves with suspected BRD, yielding 492 M. haemolytica and 215 P. multocida isolates. These isolates underwent antimicrobial susceptibility testing and screening for oxytetracycline resistance genes. RESULTS:Over 1 y post-oxytetracycline withdrawal, minimum inhibitory concentration distributions for oxytetracycline indicated a significant shift towards susceptibility in both species; resistance to oxytetracycline declined from 9.2% to 0% in M. haemolytica and from 75.0% to 5.6% in P. multocida. The minimum inhibitory concentrations of florfenicol and macrolides (tilmicosin and tulathromycin) decreased with time. Most tetracycline-resistant isolates carried tet(H). After oxytetracycline discontinuation, the BRD incidence increased, and average daily gain decreased; however, both improved following the introduction of an inactivated trivalent BRD vaccine. CONCLUSIONS:Discontinuing long-term prophylactic antimicrobial administration can restore antimicrobial susceptibility in BRD pathogens and aid in preserving therapeutic antimicrobial effectiveness when implemented alongside vaccination strategies.