We study the fatigue life and microstructure of railway axles with certain specific features of macrostructure detected with the help of ultrasonic testing (axle No. 178) and without these features (axle No. 143) of EA1N grade produced according to the EN 13261:2020 standard. We perform the comparative analysis of the corresponding samples taken near the surface at a distance of a half of radius and in the central part of the analyzed railway axles. It is shown that the fatigue life of the metal in the surface layer of the investigated axles is higher than for the metal at a depth of one half of radius of the axles and in its central part. Moreover, the difference between the detected values over the cross section of the axles with defects does not exceed 10%, whereas for the axle without defects the value of fatigue life of the metal near the surface is almost five times greater than for the samples cut out from the central part. According to the results of microstructural and fractographic analyses, we establish the direct dependence between the initiation of fatigue cracks and the size and number of nonmetallic inclusions and micropores formed in steel and its structural inhomogeneity.