The present study aimed at strengthening prior austenite grain boundary (PAGB) cohesive energy using carbon segregation and investigated the effect of carbon segregation at PAGB on the microscopic crack propagation behavior of hydrogen-related intergranular fractures in high-strength martensitic steels. At the low hydrogen content (below 0.2 wt. ppm), the fracture initiation toughness (JIC) and tearing modulus (TR), corresponding to crack growth resistance, were significantly improved by carbon segregation. In contrast, JIC and TR did not change by carbon segregation at the high hydrogen content (above 0.5 wt. ppm). Considering the non-linear relationship between the toughness properties and the PAGB cohesive energy, the experimentally evaluated toughness properties (JIC and TR) and the GB cohesive energy previously calculated by first-principles calculations were semi-quantitatively consistent even at the high hydrogen content. The microstructure observation confirmed that the plastic deformation associated with crack propagation, such as the local ductile fracture of uncracked ligaments and the formation of dislocation cell structures / nano-voids, played an important role in the non-linear relationship between the toughness properties and PAGB cohesive energy.