Fatigue design of road bridges according to Eurocode standards can be based on damage accumulation, provided that information about loading spectrum is available. Characteristic values of fatigue resistance and load effects as well as partial safety factors are introduced in the design equation in order to achieve a target reliability level. Partial safety factors are used to obtain the fatigue design S-N curves from the characteristic S-N curves. New probabilistic framework proposed by the authors for calibration of fatigue partial safety factors allows for a redefinition of Eurocode-based design S-N curves. The new framework improves the confidence in the reliability level represented by the design S-N curves. In this paper a real case study showing the impact of re-calibration of design S-N curves on fatigue design of a road bridge is presented. Importance of choice of design S-N curves is assessed both in terms of allowable traffic volume and of bridge fatigue design life.