Generally, the fatigue strength of ultra-high strength steel (UHSS) and high strength steel (HSS) arc-welded joints are comparable regardless of base metal's strength. Still, the micro-needle peening (MNP) method can improve the fatigue strength to the level of those of base metals. To understand the mechanism of this improvement, this paper investigates the microstructure of UHSS (tensile stress grade of 980 MPa) arc-welded joints treated with MNP and compares it to HSS (tensile stress grade of 440 MPa) joints. We focus on the presence of nanotwins, which exhibited a minimum thickness of 4.7 nm, observed in the UHSS joints following the MNP treatment. Importantly, these nanotwins demonstrated remarkable stability even under cyclic loading conditions (nominal stress σn = 600 MPa, N = 3 × 106 cycles). This indicates that the nanotwins contribute to the significant improvement in fatigue strength demonstrated by MNP. However, the nanotwins were not observed in the HSS joints, suggesting sufficient driving stress is necessary for their occurrence. The dislocation pileup stress at the grain boundary during twinning was estimated by the thickness of the twin, which was 8.1 GPa. This value is of the same order of magnitude as the 3.7 GPa estimated by the Hall-Petch coefficient for ferritic steel. The lower levels of C, Si, and Mn can contribute to the lower pileup stress, resulting in absence of the nanotwins in the 440 MPa joint. Overall, this study provides insights into the microstructural changes induced by MNP treatment and their impact on the fatigue strength.