In this study, high-cycle S-N characteristics and fatigue cracking behaviors under near-yield mean stress of 42CrMo steel with two different fine/coarse-grain tempered sorbite/bainite microstructures (FGM/CGM) were investigated. The results show that fatigue strength is greater for FGM than for CGM as expected, but fatigue crack growth (FCG) threshold is higher for the latter. Compared to FGM with a single crack source, CGM has multiple fatigue sources together with higher-degree ratchetting-fatigue damage, i.e., plastic-strain, dislocation cells and sub-grains, thus accelerating fatigue crack initiation. In the near-threshold regime, roughness-induced crack closure in coarse-microstructures of CGM leads to FCG retardation behavior, which increases FCG threshold. In the Paris regime, CGM presents a similar FCG rate to FGM. Microstructure barriers (i.e., crystal interfaces, carbides and interfaces with different orientations), secondary cracks, crack-path fluctuations, ratchetting and plastic strain jointly result in severer crack-tip stress shielding effects in CGM than in FGM. However, fatigue crack tends to propagate through the high Schmid factor region with {110}<111> slip system with lower slip resistance in FGM but not in CGM, which facilitates fatigue cracking of the former. Under the combined effects of the above factors, similar FCG rates are thus exhibited in both steels with different tensile strengths.