GPCR functional selectivity opens new opportunities for the design of safer drugs. Ligands orchestrate GPCR signaling cascades by modulating the receptor conformational landscape. Our study provides insights into the dynamic mechanism enabling opioid ligands to preferentially activate the G protein over the beta-arrestin pathways through the mu-opioid receptor (mu OR). We combine functional assays in living cells, solution NMR spectroscopy, and enhanced-sampling molecular dynamic simulations to identify the specific mu OR conformations induced by G protein-biased agonists. In particular, we describe the dynamic and allosteric communications between the ligand-binding pocket and the receptor intracellular domains, through conserved motifs in class A GPCRs, Most strikingly, the biased agonists trigger mu OR conformational changes in the intracellular loop 1 and helix 8 domains, which may impair beta-arrestin binding or signaling. The findings may apply to other GPCR families and provide key molecular information that could facilitate the design of biased ligands.