Although there have been substantial advances in our knowledge of the resistance of diffuse large B cell lymphoma (DLBCL) to chemotherapy, there are few efficient treatment strategies for recurrent/refractory DLBCL. The aim of this study was to investigate the role of aldehyde dehydrogenase (ALDH) 1A1 in the resistance of diffuse large B cell lymphoma to the chemotherapeutic mixture consisting of cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP). The involvement of ALDH1A1 in DLBCL was elucidated by knockdown and pharmacologic inhibition; Cell Counting Kit-8 (CCK-8) and clone formation assays were used to determine its role in CHOP sensitivity and clone formation ability. Caspase colorimetric assay was used to measure the extent of apoptosis. Western blot analysis was used to measure signal transducer and activator of transcription 3 (STAT3)/nuclear factor kappa B (NF-κB) signaling proteins, and quantitative real-time PCR (RT-PCR) was used to measure the differential expression of ALDH1A1 of DLBCL patients and healthy donors. ALDH1A1 showed a 5.64-fold higher expression in malignant B cells than in normal B cells. Diethylaminobenzaldehyde (DEAB) decreased the half maximal inhibitory concentration (IC50) of the CHOP regimen in Farage cells from 344.78 ± 65.75 to 183.88 ± 49.75 ng/ml (P = 0.004). Both knockdown and inhibition of ALDH1A1 reduced clonogenicity, increased caspase-3/caspase-9 activity, and attenuated the phosphorylation status of STAT3/NF-κB. The prognosis of patients with a high level of ALDH1A1 expression was poor compared with that of patients with low levels of expression (P = 0.044). ALDH1A1 is a new mediator for resistance of DLBCL to CHOP; it is a predictor of clinical prognosis and may serve as a potential target to improve chemotherapy responsiveness of human DLBCL.