The ATP-binding cassette (ABC) F/GCN (general control non-derepressible) subfamily is a group of soluble ABC proteins that has two nucleotide binding domains (NBDs) but no transmembrane domains (TMDs). Previous study has indicated that ABCF3, also called GCN20/SCORD5, is involved in stress-associated protein translation control and defense responses to bacterial infection. Here, we show that ABCF3 regulates H2O2 uptake and endoplasmic reticulum (ER) stress responses in Arabidopsis. ABCF3 expressed mainly in leaves, stems, root tips, seeds, and anther. Analysis of abcf3 mutants and transgenic overexpressors (OX) of ABCF3 indicates that ABCF3 regulates H2O2 uptake. qRT-PCR analysis indicated that ABCF3 affects the expression of several aquaporin genes that were involved in H2O2 uptake in plants. In addition, abcf3 mutants exhibited enhanced sensitivity to ER stress and amino acid deprivation when challenged with ER stress inducers tunicamycin and amino acid synthesis inhibitor chlorsulfuron compared to wild type. Overexpression of AtABCF3 increases, whereas, abcf3 mutants depress the expression of ER stress-related genes. Taken together, these results indicated that ABCF3 regulates stress response by modulating aquaporin gene expression and ER stress responses.