One of the main challenges of sustainable agriculture is improving food production while reducing significant impact on the soil, water, and other environmental resources. In this context, the use of humic substances extracted from different substrates in agricultural practices has been envisioned as a promising nature-like and environmental-friendly technology to support crop yield and quality. Humic substances, deriving from chemical and biological transformations of biota materials, represent an intrinsic component of soil organic matter (SOM) consisting of associations of relatively small humic molecules linked together through hydrophobic interactions and hydrogen bonds. Because of their distinctive physicochemical features, they are used in several industrial and agricultural applications and in remediation technologies for metal-contaminated soils. Humic substances are of pivotal importance for environmental protection by conditioning soils and improving their stability and resistance to erosion. In addition, they possess inherent hormone-like nature and exhibit biological activity. This is often associated with complementary action of soil microbiota and is manifested in their capacity to modulate the transport and bioavailability of nutrients to plants, influence root growth and architecture, enhance crop yields and regulate the expression of a broad array of genes involved in plant metabolism, development and resistance to stress. Despite significant efforts to explain the molecular structure of humic substances and its relationship with a plurality of physiological responses and signalling networks triggered in plants, several functional aspects still need to be clarified. One major issue is that humic substances possess a very complex structure, which accounts for their multifaceted biological action. Therefore, this Research Topic aims to update the knowledge on humic substances by improving the current understanding of their structure and interactions with plants and associated rhizosphere microorganisms, thus shining light on the mechanisms and cellular signalling pathways through which humic substances target specific plant metabolic routes and elicit physiological responses. Implications of such interactions are expected to be assessed using differential methodological approaches, under either small scale trials or field conditions, in view of developing advanced and sustainable agriculture technologies aimed at improving crop yield and food quality.