Abstract <p><i>Cannabis sativa</i> L. roots have been less studied than aboveground organs, despite their key role in plant physiology, metabolism, and interactions with biotic and abiotic factors. Metabolomic and phytochemical analyses reveal that roots synthesize a diverse array of bioactive compounds with antimicrobial, anti-inflammatory, antioxidant, and cytotoxic properties, highlighting their biotechnological potential. Root exudation patterns and interactions with endophytic microorganisms modulate rhizosphere microbial networks that support nutrient uptake, stress tolerance, pathogen resistance, and whole-plant physiology. Root-derived phytohormones and other signalling molecules may participate in coordinating biochemical pathways between belowground and aboveground tissues, with potential effects on secondary metabolism in aerial tissues. Recent advances in metabolomics, transcriptomics, microfluidic rhizosphere systems, and root-specific genetic engineering now enable detailed investigation of root metabolism in <i>Cannabis sativa</i> L. This review synthesises current knowledge on the metabolic roles of <i>Cannabis sativa</i> L. roots, their interactions with the rhizosphere microbiome, and root-derived systemic signalling. It emphasises aspects of root biology that are central to fundamental plant processes and to the development of sustainable strategies for optimising phytochemical yields. By placing roots at the forefront, this synthesis underscores the need to expand research beyond aerial tissues to fully understand and harness the biotechnological potential of <i>Cannabis</i> species.</p> Key points <p>• <i>Root metabolism and signalling regulate whole-plant-metabolic pathways</i></p> <p>• <i>Root-associated microbiomes influence nutrient dynamics and phytochemical profiles</i></p> <p>• <i>Root culture systems provide a scalable platform for biotechnological manipulation aimed at the production of bioactive compounds.</i></p>