High-cycle fatigue properties of medium carbon microalloyed (MA) steel 38MnVS with different microstructural characteristics were studied by rotating bending fatigue test. Low alloy steel 40Cr with quenched and tempered (QT) microstructure was also used for comparison. The results show MA steel in the as-forged condition has the best fatigue property mainly owing to the significant precipitation strengthening of fine V(C,N) precipitates and much fine ferrite–pearlite microstructure, which is even better than that of the QT steel 40Cr. The formation of film-like ferrite along coarse prior austenite grain boundary and coarse bainitic type microstructure as well as the coarsening of the initial fine precipitates significantly deteriorated the MA steel's fatigue properties. SEM examination of fatigue fracture surfaces revealed that the fatigue fracture mechanism of MA steel is different from that of QT steel. It is concluded that microstructure of ferrite–pearlite type MA steels should be carefully controlled after rolling and/or forging to obtain improved fatigue properties.