Hydrogen-induced cracking (HIC) susceptibility tests, hydrogen permeation tests and the electron backscatter diffraction (EBSD) measurements were used to study the effect of Nb on the HIC resistance of the high-strength low-alloy (HSLA) steel. The Nb microalloying significantly improved the HIC resistance and decreased the proportion of intergranular cracks through the hydrogen trapping effect and microstructure optimization. Dispersed nanometer-sized NbC precipitates effectively increased the number of irreversible traps, hindered hydrogen aggregation, and enhanced the resistance to crack initiation and propagation. Moreover, Nb optimized the microstructure by increasing the proportion of the low-angle grain boundaries (LAGBs), refining the prior austenite grain size, decreasing the dislocation density, and changing the distribution of Σ3 boundaries.