Oxidative stress caused by cellular accumulation of reactive oxygen species (ROS) is a major contributor to disease and cell death. However, how induced pluripotent stem cells (iPSC) respond to different levels of oxidative stress is largely unknown. Here, we investigated the effect of H2O2‐induced oxidative stress on iPSC function in vitro. Mouse iPSC were treated with H2O2 (25–100 μmol/L). IPSC adhesion, migration, viability, apoptosis and senescence were analysed. Expression of adhesion‐related genes, stress defence genes, and osteoblast‐ and adipocyte‐associated genes were determined by reverse transcription polymerase chain reaction. The present study found that H2O2 (25–100 μmol/L) decreased iPSC adhesion to matrix proteins and endothelial cells, and downregulated gene expression levels of adhesion‐related molecules, such as integrin alpha 7, cadherin 1 and 5, melanoma cell adhesion molecule, vascular cell adhesion molecule 1, and monocyte chemoattractant protein‐1. H2O2 (100 μmol/L) decreased iPSC viability and inhibited the capacity of iPSC migration and transendothelial migration. iPSC were sensitive to H2O2‐induced G2/M arrest, senescence and apoptosis when exposed to H2O2 at concentrations above 25 μmol/L. H2O2 increased the expression of stress defence genes, including catalase, cytochrome B alpha, lactoperoxidase and thioredoxin domain containing 2. H2O2 upregulated the expression of osteoblast‐ and adipocyte‐associated genes in iPSC during their differentiation; however, short‐term H2O2‐induced oxidative stress did not affect the protein expression of the pluripotency markers, octamer‐binding transcription factor 4 and sex‐determining region Y‐box 2. The present results suggest that iPSC are sensitive to H2O2 toxicity, and inhibition of oxidative stress might be a strategy for improving their functions.