Limited research exists on impacts of air pollution on non-human mammals, particularly animal athletes such as Thoroughbred racehorses. Athletes have a greater risk of exposure as heightened exertion and increased airflow carry more pollutants deeper into the respiratory tract. To provide insights into the impact of ambient air pollution, particularly fine particulate matter (PM2.5), on race speed. Retrospective observational study. Data were obtained from The Jockey Club Information Systems, covering 31 407 winning races by Thoroughbred horses in California spanning 10 years (2011-2020) and evaluated the association between air pollution and winning race speeds. For race days, we collected PM2.5 data from the nearest U.S. Environmental Protection Agency (EPA) monitoring site within 100 km of each racetrack (n = 12). We assessed the associations between daily average PM2.5 concentrations and speed of winning horses with linear mixed effects regression. We adjusted for horse characteristics, race-related covariates, temporal indicators (e.g., year), other air pollutants and temperature. We conducted sensitivity analyses by adjusting extreme air pollution days by reassigning values to the 95th percentile value and conducting linear mixed effects regression on series of datasets with incremental cutpoints of PM2.5. In the cutpoint analysis, we found that for PM2.5 between 4 and 23.6 μg/m3, speed decreased 0.0008 m/s (95% CI: -0.0014562 to -0.00018) for every 1 μg/m3 increase of PM2.5. Limitations include the use of offsite monitors leading to imprecise exposure measurements, not using training practice data, and generalisability as the study focuses on California racetracks. This study highlights the need to create advisories to safeguard the performance of horses during periods of poor air quality. Further research is recommended to explore additional factors influencing the relationship between air pollution and equine welfare.