<p>This study investigates the influence of Ni content on the microstructure and mechanical properties of EH40 steel welded by keyhole TIG welding. EH40 steel is widely used in polar environments due to its high strength and low-temperature toughness, but optimizing weld properties remains a challenge. Ni sheets of varying thicknesses were introduced to control the Ni content in the weld. The microstructure was characterized using optical microscopy, X-ray diffraction, energy-dispersive spectroscopy, and electron backscatter diffraction. Mechanical properties were evaluated through tensile testing, microhardness measurements, and low-temperature impact toughness tests. The results indicate that a moderate Ni content (3.35 wt.%) refines the acicular ferrite structure, enhances the proportion of high-angle grain boundaries, and significantly improves low-temperature toughness, with impact energy absorption increasing to 67 J—an improvement of 644% compared to Ni-free welds. Weld microhardness also increases, reaching 333.1 HV. Excessive Ni content (&gt;7.3 wt.%) leads to grain coarsening, reduced toughness, and lower hardness due to complex phase transformations. These findings highlight that controlling Ni content within the range of 3.35–7.3 wt.% optimizes both microstructure and mechanical performance, providing valuable guidance for the application of EH40 steel in polar environments.</p>