Cardiovascular mortality has been shown to increase in the winter. However, it is unclear whether cold temperature affects indicators known as cardiovascular markers. We evaluated the association between ambient temperature and cardiovascular markers using data collected retrospectively from 55,567 adults who had visited a health check-up clinic between 1995 and 2008. Non-parametric smoothing regressions were fitted to determine the shapes of association between temperature and cardiovascular markers such as blood pressure, lipid profiles, platelet count, and high sensitivity C-reactive protein (hsCRP). Mixed effect model was used to investigate the significance of the relationship between temperature and cardiovascular markers. Decreased ambient temperature was associated with an increase in systolic and diastolic blood pressures, platelet count and serum low density lipoprotein-cholesterol concentration. In contrast, high density lipoprotein-cholesterol level declined with decreasing temperature. The hsCRP level increased with decreasing temperature in the minimum temperatures below 0°C, but revealed a reverse association above. Our study suggests that excess cardiovascular mortality in cold weather might be associated with temperature-related variations of cardiovascular markers.