Microstructural differences through the thickness have always been an attention focus for large-scale steel structures, since they could significantly influence fatigue crack propagation (FCP) characteristics. Here, the effects of microstructure in FCP behaviors of 42CrMo steel were analyzed with emphasis on two different tempered sorbite/bainite (TS/B) microstructures: (i) fine TS/B structures (M42); (ii) coarse TS/B structures (M52). The results show that the FCP rate is relatively slower for M52 than for M42 in the regime through the later stage of near-threshold (I-L) to the early stage of Paris-regime (II-E) with low AK, but is fairly similar for the two cases in the other AK regimes. Under relatively low AK, the FCP path of M52 is more tortuous with numerous micro-cracks in B and larger-sized crack deflections at the carbides and boundaries compared to M42, especially in the I-L where fatigue micro-cracks can deflect at the single coarse carbide, thereby resulting in roughness-induced crack closure and crack retardation behavior. Theoretical analysis shows that FCP behaviors are closely related to the cyclic plastic region size (Arp) at the crack-tip. In the early stage of near-threshold region, Arp is restricted within a grain-size scale for both steels, thus leading to a similar FCP rate. However, from the I-L to the II-E, coarse B/ferrite-grains in M52 are always larger than the Arp of fatigue crack-tip, and thereby lead to numerous micro-cracks formation and crack deflections at the carbides and B boundaries, inducing crack closure. In the later stage of Paris region, B/ferrite-grains in the two cases are much smaller than Arp, so that FCP behaviors depend on the microstructure group effects, or overall strength and fracture toughness of materials, which cause the close FCP rate.