An attempt has been made to systematically investigate the effects of microstructural parameters, such as the prior austenite grain size (PAGS), in influencing the resistance to fatigue crack growth (FCG) in the near-threshold region under three different temper levels in a quenched and tempered high-strength steel. By austenitizing at various temperatures, the PAGS was varied from about 0.7 to 96 μm. The microstructures with these grain sizes were tempered at 200 °C, 400 °C, and 530 °C and tested for fatigue thresholds and crack closure. It has been found that, in general, three different trends in the dependence of both the total threshold stress intensity range, ΔK th , and the intrinsic threshold stress intensity range, ΔK eff, th , on the PAGS are observable. By considering in detail the factors such as cyclic stress-strain behavior, environmental effects on FCG, and embrittlement during tempering, the present observations could be rationalized. The strong dependence of ΔK th and ΔK eff, th on PAGS in microstructures tempered at 530 °C has been primarily attributed to cyclic softening and thereby the strong interaction of the crack tip deformation field with the grain boundary. On the other hand, a less strong dependence of ΔK th and ΔK eff, th on PAGS is suggested to be caused by the cyclic hardening behavior of lightly tempered microstructures occurring in 200 °C temper. In both microstructures, crack closure influenced near-threshold FCG (NTFCG) to a significant extent, and its magnitude was large at large grain sizes. Microstructures tempered at the intermediate temperatures failed to show a systematic variation of ΔKth and ΔKeff, th with PAGS. The mechanisms of intergranular fracture vary between grain sizes in this temper. A transition from “microstructure-sensitive” to “microstructure-insensitive” crack growth has been found to occur when the zone of cyclic deformation at the crack tip becomes more or less equal to PAGS. Detailed observations on fracture morphology and crack paths corroborate the grain size effects on fatigue thresholds and crack closure.