Background Controversy remains regarding the relationship between ambient ozone and mortality worldwide. In mainland China, the largest developing country, there has been no prior study investigating the acute effect of O3 on death risk. Given the changes in types of air pollution from conventional coal combustion to the mixed coal combustion/motor vehicle emissions in China’s large cities, it is worthwhile to investigate the acute effect of O3 on mortality outcomes in the country. Objectives We conducted a time-series study to investigate the relation between O3 and daily mortality in Shanghai using 4 years of daily data (2001–2004). Methods We used the generalized additive model with penalized splines to analyze mortality, O3 pollution, and covariate data in warm and cold seasons. We considered daily counts of all-cause mortality and several cause-specific subcategories (respiratory and cardiovascular). We also examined these associations among several subpopulations based on age and sex. Results O3 was significantly associated with total and cardiovascular mortality in the cold season but not in the warm season. In the whole-year analysis, an increase of 10 μg/m3 of 2-day average (lag01) O3 corresponds to 0.45% [95% confidence interval (CI), 0.16–0.73%], 0.53% (95% CI, 0.10–0.96%), and 0.35% (95% CI, −0.40 to 1.09%) increase of total nonaccidental, cardiovascular, and respiratory mortality, respectively. In the cold season, the estimates increased to 1.38% (95% CI, 0.68–2.07%), 1.53% (95% CI, 0.54–2.52%), and 0.95% (95% CI, −0.71 to 2.60%), respectively. In the warm season, we did not observe significant associations for both total and cause-specific mortality. The results were generally insensitive to model specifications such as lag structure of O3 concentrations and degree of freedom for time trend. Multipollutant models indicate that the effect of O3 was not confounded by particulate matter ≤ 10 μm in diameter (PM10) or by sulfur dioxide; however, after adding nitrogen dioxide into the model, the association of O3 with total and cardiovascular mortality became statistically insignificant. Conclusions O3 pollution has stronger health effects in the cold than in the warm season in Shanghai. Our analyses also strengthen the rationale for further limiting levels of O3 pollution in outdoor air in the city.