SummaryThe combination of β‐oligosaccharides from enzymatically hydrolysed barley β‐glucan has attracted interest recently due to its positive effects on human health. This study aimed to assess the impact of the endo‐β‐1,3‐1,4‐glucanase enzyme from Bacillus subtilis 168 on improving the nutritional and bioactive properties of barley β‐glucan. A new procedure for the isolation of β‐glucan was developed, at a lower temperature (45 °C), enabling purity from starch contamination, without affecting the yield (6 g β‐glucan from 100 g of barley flour). The endo‐β‐1,3‐1,4‐glucanase is cloned into E. coli pQE_Ek enables the high production and purification (82% yield, 1.8 mg mL⁻¹ and 440 U mg⁻¹) of an enzyme identical to the natural one (25.5 kDa). The enzymatic reaction showed high efficiency of β‐glucan degradation by recombinant enzyme, giving a mixture of products (of which 3‐O‐β‐cellobiosyl‐D‐glucose and 3‐O‐β‐cellotriosyl‐D‐glucose are the most abundant), the reduction of viscosity (17%) and increase in antioxidant capacities by 15.2%, 30.9% and 44.0% assessed by ABTS, DPPH and ORAC, respectively. These results indicate the possible application of endo‐β‐1,3‐1,4‐glucanase enzyme in improving the properties of barley β‐glucan used as functional foods.