While mobile genetic elements (MGEs) critically influence antibiotic resistance gene (ARG) dissemination, the regulatory role of bacteriophages as unique MGEs remains enigmatic in natural ecosystems. Through a global-scale phage-resistome interrogation spanning 840 groundwater metagenomes, we established a large aquifer resistome repository and uncovered three paradigm-shifting discoveries. First, phages harboured markedly fewer ARGs compared to plasmids and integrative elements, but their bacterial hosts paradoxically maintained the highest anti-phage defence gene inventories, showing an evolutionary equilibrium where investment in phage defence constrains ARG acquisition. Second, lytic phages demonstrated dual functionality characterized with directly suppressing ARG transmission through host lysis while indirectly enriching defence genes that inhibit horizontal gene transfer. Third, vertical inheritance sustained ARGs in 11.2% of MGE-free groundwater microbes. We further extended linkages between ARG profiles, phage defences and biogeochemical genes, revealing phage-mediated co-occurrence of ARGs and denitrification genes in shared hosts. These findings pioneer a phage-centric framework for resistome evolution, guiding phage-based ARG mitigation in groundwater ecosystems.
