To understand the changes in arabinoxylan (AX) during breadmaking, multi-step enzyme digestion was conducted to re-extract arabinoxylan (AX-B) from AX-fortified bread. Their structural changes were compared using HPSEC, HPAEC, FT-IR, methylation analysis, and 1H NMR analysis; their properties changes in terms of enzymatic inhibition activities and in vitro fermentability against gut microbiota were also compared. Results showed that AX-B contained a higher portion of covalently linked protein while the molecular weight was reduced significantly after breadmaking process (from 677.1 kDa to 15.6 kDa); the structural complexity of AX-B in terms of the degree of branching was increased; the inhibition activity against α-amylase (76.81 % vs 73.89 % at 4 mg/mL) and α-glucosidase (64.43 % vs 58.08 % at 4 mg/mL) was improved; the AX-B group produced a higher short-chain fatty acids concentration than AX (54.68 ± 7.86 mmol/L vs 44.03 ± 4.10 mmol/L). This study provides novel knowledge regarding the structural and properties changes of arabinoxylan throughout breadmaking, which help to predict the health benefits of fibre-fortified bread and achieve precision nutrition.