•Alcalase-hydrolyzed soybean hydrolysate displayed chemical antioxidant activities.•SPH decreased ROS levels in Caco-2 cells under both normal and stress conditions.•In vitro GI digestion enhanced the antioxidant activities of SPH in general.•Transepithelial transport of SPH was studied and absorbed peptides were identified. Peptides from Alcalase-hydrolyzed soybean protein hydrolysate (SPH) may hold the potential as natural antioxidants. In addition, the effect of human gastrointestinal (GI) tract on peptide bioavailability needs to be explored. In this study, the impact of simulated GI digestion and transepithelial transport on various antioxidant properties of SPH were investigated. SPH displayed DPPH radical scavenging (IC50 = 4.22 mg/mL), ABTS+ radical scavenging (IC50 = 2.93 mg/mL), reducing power and metal ion-chelating activities (IC50 = 0.67 mg/mL). Furthermore, SPH significantly (P < .05) inhibited the generation of intracellular reactive oxygen species (ROS) in Caco-2 cells. After simulated GI digestion, the antioxidant properties of SPH were enhanced, except for a decrease in ABTS+ radical scavenging activity. After transepithelial transport, the permeates maintained partial antioxidant activity and the LC-MS/MS data further identified the absorbed soybean peptides. These results suggest that SPH contains the antioxidant peptides that are potentially bioavailable and can be regarded as a promising source of functional food ingredients.