To investigate the effect of oat bran on bread quality and the mechanism of reducing the glycemic index (GI) of bread, wheat bran (10%, w/w, flour basis), oat bran (10%), and β-glucan (0.858%) were individually added to determine the expansion of dough, the specific volume, texture, color, GI, starch digestion characteristics, and α-amylase inhibition rate of bread. The results showed that the incorporation of wheat bran and oat bran both reduced the final expanded volume of the dough, decreased the specific volume of the bread, and increased the bread hardness and crumb redness and greenness values as compared to the control wheat group. The above physical properties of bran-containing bread obviously deteriorated while the bread with β-glucan did not change significantly (p < 0.05). The GI in vitro of bread was in the following order: control (94.40) > wheat bran (69.24) > β-glucan (65.76) > oat bran (64.93). Correspondingly, the oat bran group had the highest content of slowly digestible starch (SDS), the β-glucan group had the highest content of resistant starch (RS), and the control group had the highest content of rapidly digestible starch (RDS). For the wheat bran, oat bran, and β-glucan group, their inhibition rates of α-amylase were 9.25%, 28.93%, and 23.7%, respectively. The β-glucan reduced the bread GI and α-amylase activity by intertwining with starch to form a more stable gel network structure, which reduced the contact area between amylase and starch. Therefore, β-glucan in oat bran might be a key component for reducing the GI of whole oat bread.