Nickel (Ni)—a component of urease and hydrogenase—was the latest nutrient to be recognized as an essential element for plants. However, to date there are no records of Ni deficiency for annual species cultivated under field conditions, possibly because of the non-appearance of obvious and distinctive symptoms, i.e., a hidden (or latent) deficiency. Soybean, a crop cultivated on soils poor in extractable Ni, has a high dependence on biological nitrogen fixation (BNF), in which Ni plays a key role. Thus, we hypothesized that Ni fertilization in soybean genotypes results in a better nitrogen physiological function and in higher grain production due to the hidden deficiency of this micronutrient. To verify this hypothesis, two simultaneous experiments were carried out, under greenhouse and field conditions, with Ni supply of 0.0 or 0.5 mg of Ni kg−1 of soil. For this, we used 15 soybean genotypes and two soybean isogenic lines (urease positive, Eu3; urease activity-null, eu3-a, formerly eu3-e1). Plants were evaluated for yield, Ni and N concentration, photosynthesis, and N metabolism. Nickel fertilization resulted in greater grain yield in some genotypes, indicating the hidden deficiency of Ni in both conditions. Yield gains of up to 2.9 g per plant in greenhouse and up to 1,502 kg ha−1 in field conditions were associated with a promoted N metabolism, namely, leaf N concentration, ammonia, ureides, urea, and urease activity, which separated the genotypes into groups of Ni responsiveness. Nickel supply also positively affected photosynthesis in the genotypes, never causing detrimental effects, except for the eu3-a mutant, which due to the absence of ureolytic activity accumulated excess urea in leaves and had reduced yield. In summary, the effect of Ni on the plants was positive and the extent of this effect was controlled by genotype-environment interaction. The application of 0.5 mg kg−1 of Ni resulted in safe levels of this element in grains for human health consumption. Including Ni applications in fertilization programs may provide significant yield benefits in soybean production on low Ni soil. This might also be the case for other annual crops, especially legumes.