To study the fatigue properties of HRB500 reinforcing bar in longitudinal connected slab track on bridge, fatigue tests under symmetric axial alternating tension-compression cyclic loads for standard specimens of HRB500 reinforcing bar were conducted, and then the fatigue life and its cumulative failure probability of standard HRB500 reinforcing bar at different stress levels were obtained. On this basis, the correlation coefficient p and Theil's inequality coefficient u were used as indexes of estimation accuracy to evaluate three methods which are widely used to estimate parameters of three-parameter Weibull distribution. And the grey prediction method, whose prediction accuracy is the highest among the three methods, was used to estimate the parameters of three-parameter Weibull distribution for the fatigue life of standard HRB500 reinforcing bar, and the distribution functions for the fatigue life of HRB500 reinforcing bar at different stress levels were obtained. Ultimately, the probabilistic S-N curve of HRB500 reinforcing bar in the longitudinal connected slab track on bridge with reliable index of 4.2 based on Kohout-Vêchet equation in the full stress range was established. The research can provide experimental basis for the establishment of fatigue life prediction model of HRB500 reinforcing bar in the longitudinal connected slab track on bridge under combined loads during service life.