The objective of this study was to investigate the effect of β-glucan on the pasting, gelling, rheological properties, and multi-level structures of the highland barley (HB) starch after dynamic high pressure microfluidization (DHPM) treatment, exploring the inhibition mechanisms of starch retrogradation by endogenous β-glucan after DHPM. DHPM treatment led to a decrease in the viscosity (K values from 161.1 to 54.4) of HB starch-β-glucan composite as the pressure increased from 0 to 120 MPa, while an increase in the viscosity was induced by DHPM treatment cycles from 1 to 3 at 120 MPa. Similar changes were also found in the relative crystallinity (RC) and degree of retrogradation (DR). The RC values decreased from 44.66 % to 25.53 %, and the DR values decreased from 53.4 % to 24.6 % as the DHPM pressure increased from 0 MPa to 120 MPa (p <
0.05). However, when number of DHPM cycles increased from 1 to 3 under 120 MPa, the RC value and DR value increased to 35.99 % and 31.9 %, respectively. Scanning electron microscopy images demonstrated that β-glucan formed a protective layer around HB starch granules after DHPM treatment at 120 MPa for one pass. Fourier transform infrared spectra and X-ray diffraction results indicated the intra- and intermolecular hydrogen bonds between HB starch and β-glucan were strengthened by DHPM. Endogenous β-glucan emerged as a strong candidate for inhibition of HB starch retrogradation. This study highlights an innovative and promising strategy for improving the properties of HB starch and facilitating its utilization.