Background In recent years, air pollution has become an imminent problem in China. Few studies have investigated the impact of air pollution on the mortality of the middle-aged and elderly people. Therefore, this study aims to evaluate the impact of PM2.5 (fine particulate matter) and O-3 (ozone) on non-accidental mortality and respiratory mortality of the middle-aged and elderly people in Lishui District of Nanjing and provide the evidence for potential prevention and control measures of air pollution. Method Using daily mortality and atmospheric monitoring data from 2015 to 2019, we applied a generalized additive model with time-series analysis to evaluate the association of PM2.5 and O-3 exposure with daily non-accidental mortality and respiratory mortality in Lishui District. Using the population attributable fractions to estimate the death burden caused by short-term exposure to O-3 and PM2.5?. Result For every 10 mu g/m(3) increase in PM2.5, non-accidental mortality increased 0.94% with 95% confidence interval (CI) between 0.05 and 1.83%, and PM2.5 had a more profound impact on females than males. For every 10 mu g/m(3) increase in O-3, respiratory mortality increased 1.35% (95% CI: 0.05, 2.66%) and O-3 had a more profound impact on males than females. Compared with the single pollutant model, impact of the two-pollutant model on non-accidental mortality and respiratory mortality slightly decreased. In summer and winter as opposed to the other seasons, O-3 had a more obvious impact on non-accidental mortality. The population attributable fractions of non-accidental mortality were 0.84% (95% CI:0.00, 1.63%) for PM2.5 and respiratory mortality were 0.14% (95% CI:0.01, 0.26%) for O-3. For every 10 mu g/m(3) decrease in PM2.5, 122 (95% CI: 6, 237) non-accidental deaths could be avoided. For every 10 mu g/m(3) decrease in O-3, 10 (95% CI: 1, 38) respiratory deaths could be avoided. Conclusion PM2.5 and O-3 could significantly increase the risk of non-accidental and respiratory mortality in the middle-aged and elderly people in Lishui District of Nanjing. Exposed to air pollutants, men were more susceptible to O-3 damage, and women were more susceptible to PM2.5 damage. Reduction of PM2.5 and O-3 concentration in the air may have the potential to avoid considerable loss of lives.