Nitrogen (N) and phosphorus (P) are two essential plant macronutrients that can limit plant growth by different mechanisms. We aimed to shed light on how soybean respond to low nitrogen (LN), low phosphorus (LP) and their combined deficiency (LNP). Generally, these conditions triggered changes in gene expression of the same processes, including cell wall organization, defense response, response to oxidative stress, and photosynthesis, however, response was different in each condition. A typical primary response to LN and LP was detected also in soybean, i.e., the enhanced uptake of N and P, respectively, by upregulation of genes for the corresponding transporters. The regulation of genes involved in cell wall organization showed that in LP roots tended to produce more casparian strip, in LN more secondary wall biosynthesis occurred, and in LNP reduction in expression of genes involved in secondary wall production accompanied by cell wall loosening was observed. Flavonoid biosynthesis also showed distinct pattern of regulation in different conditions: more anthocyanin production in LP, and more isoflavonoid production in LN and LNP, which we confirmed also on the metabolite level. Interestingly, in soybean the nutrient deficiencies reduced defense response by lowering expression of genes involved in defense response, suggesting a role of N and P nutrition in plant disease resistance. In conclusion, we provide detailed information on how LN, LP, and LNP affect different processes in soybean roots on the molecular and physiological levels.