Abstract: To reveal the grain boundary weakening mechanism in the heat-affected zone of martensitic heat-resistant steel welded joints, clarify the segregation behavior of boron at grain boundaries and its regulation on the mechanical properties of grain boundaries, and provide a theoretical basis for welding process optimization and crack-resistant composition design of base metals, a systematic multi-scale study combining optical microscopy, scanning electron microscopy, electron probe element characterization, first-principles calculations, and thermodynamic simulations was conducted on 9Cr-3W-3Co martensitic heat-resistant steel welded joints. The results show that boron exhibits significant segregation at grain boundaries in the coarse-grained heat-affected zone, with a segregation coefficient of 3.81. Boron atoms preferentially occupy octahedral interstitial sites in body-centered cubic iron, with a segregation energy of approximately −0.35 eV; after segregation, the hybridization between B-p orbitals and Fe-d orbitals suppresses the original metallic bonding among iron atoms, reducing the grain boundary separation work from 2.51 J/m² to 2.21 J/m² with a weakening amplitude of 11.95%. The boron content is linearly and negatively correlated with the initial melting temperature of the alloy. When the boron content exceeds a mass fraction of 0.007%, M₃B₂ borides precipitate and alter the precipitation behavior of phases such as M₂₃C₆. This study establishes a multi-scale correlation between boron segregation and grain boundary weakening, which provides a theoretical support for the welding process optimization of martensitic heat-resistant steel and the base metal composition design based on critical boron content control.