For many years, biologists have focused on the role of Pitx2, expressed on the left side of developing embryos, in governing organ laterality. Here, we identify a different pathway during left-right asymmetry initiated by the right side of the embryo. Surprisingly, this conserved mechanism is orchestrated by the extracellular glycosaminoglycan, hyaluronan (HA) and is independent of Pitx2 on the left. Whereas HA is normally synthesized bilaterally as a simple polysaccharide, we show that covalent modification of HA by the enzyme Tsg6 on the right triggers distinct cell behavior necessary to drive the conserved midgut rotation and to pattern gut vasculature. HA disruption in chicken and Tsg6−/− mice results in failure to initiate midgut rotation and perturbs vascular development predisposing to midgut volvulus. Our study leads us to revise the current symmetry-breaking paradigm in vertebrates and demonstrates how enzymatic modification of HA matrices can execute the blueprint of organ laterality. Display omitted •Left-right asymmetry is initiated by the right side of the embryo•Extracellular hyaluronan determines gut and vascular laterality•Tsg6-catalyzed hyaluronan modification on the right is independent of Pitx2 on the left•Tsg6 null mice fail to initiate gut rotation predisposing to volvulus Gut malrotation predisposes newborns to catastrophic strangulation of the intestine. Sivakumar et al. show that covalent modification of the extracellular matrix (ECM) component hyaluronan specifically on the right side of mouse and chick embryos regulates gut rotation key to intestinal morphogenesis. Disruption of modification of hyaluronan results in gut malrotation.