BACKGROUND: Antibiotic resistance leads to longer hospital stays, higher medical costs, and increased mortality. However, research into the relationship between climate change and antibiotic resistance remains inconclusive. This study aims to address the gap in the literature by exploring the association of antibiotic resistance with regional ambient temperature and its changes over time. METHODS: Data were obtained from the China Antimicrobial Surveillance Network (CHINET), monitoring the prevalence of carbapenem-resistant Acinetobacter baumannii (CRAB), Klebsiella pneumoniae (CRKP) and Pseudomonas aeruginosa (CRPA) in 28 provinces/regions over the period from 2005 to 2019. Log-linear regression models were established to determine the association between ambient temperature and antibiotic resistance after adjustment for variations in socioeconomic, health service, and environmental factors. FINDINGS: A 1 °C increase in average ambient temperature was associated with 1.14-fold increase (95%-CI [1.07-1.23]) in CRKP prevalence and 1.06-fold increase (95%-CI [1.03-1.08]) in CRPA prevalence. There was an accumulative effect of year-by-year changes in ambient temperature, with the four-year sum showing the greatest effect on antibiotic resistance. Higher prevalence of antibiotic resistance was also associated with higher antibiotic consumption, lower density of health facilities, higher density of hospital beds and higher level of corruption. INTERPRETATION: Higher prevalence of antibiotic resistance is associated with increased regional ambient temperature. The development of antibiotic resistance under rising ambient temperature differs across various strains of bacteria. FUNDING: The National Key R&D Program of China (grant number: 2018YFA0606200), National Natural Science Foundation of China (grant number: 72074234), Fundamental Scientific Research Funds for Central Universities, P.R. China (grant number: 22qntd4201), China Medical Board (grant number: CMB-OC-19-337).