Arabinoxylan (BIF-60) was isolated from barley water-insoluble fiber (BIF) by ethanol precipitation at 60 % (v/ v). BIF-60 was composed of xylose (48.5 %) and arabinose (30.3 %). Its average molecular weight was 1360 kDa. Methylation and 1D/2D NMR analysis showed that BIF-60 possessed beta-(l -> 4)-xylan as backbone, comprised of un-substituted (1,4-linked beta-Xylp, 56.9 %), mono-substituted (1,2,4-linked and 1,3,4-linked beta-Xylp, 22.1 %) and di-substituted (1,2,3,4-lin4ked beta-Xylp, 18.4 %) xylose units, as well as other residues (T-Araf-(1 ->, T-Xylp ->(1 ->,-> 5)-> Araf-(1k ->,-> 2)-Araf-(1 ->, -> 3)-Araf-(1 -> and -> 4)-Glcp-(1 ->). BIF-60 exhibited shear-thinning behaviour, low gel stability and weak gelling ability at high concentrations. This work provides a theoretical and experimental basis for molecular structure and properties of the alkali-extracted arabinoxylan from barley kernel, which could guide further functional research and application of barley-derived arabinoxylan.