Abstract: Orthotropic steel decks have been widely used in long-span truss girder and box girder cable-stayed bridges and arch bridges duo to their good integrity and high bearing capacity, while the fatigue characteristics of railway orthotropic steel deck are significantly different from those of highway bridges. According to the fatigue-prone details in rib-to-diaphragm joints of a railway steel bridge, the local stress properties were investigated by using elastic support beam theory and closed thin-walled bar theory. Furthermore, the analytic formula for calculating the fatigue stress of fatigue-prone details in the stiffener was presented. Then the influence degree and action mechanism of various fatigue factors included in analytic formula were also analyzed. Based on the orthotropic steel deck in Yongjiang Great Bridge, a large span railway cable-stayed bridge with steel box girders, a full-scale fatigue test model including 2 U-ribs and 2 V-ribs was designed, and 5.6 million cycles of fatigue test were performed. The results revealed that the analytic formula is in good agreement with finite element analysis and test results. The experiment model could accurately simulate the stress of the fatigue sensitive point in a practical structure, and analytic theory can reflect the influence factors and rules. The weld length of rib-to-diaphragm connections and the inclination of rib plate can effectively reduce the fatigue stress of a rib when exposed to train load. The V-rib has better anti-fatigue performance than the U-rib in a railway orthotropic steel deck when the area and the bending stiffness of two types of rib scheme keep the same.