Small ubiquitin-like modifier (SUMO)/sentrin-specific protease 1 (SENP1), a member of the SENP family, is highly expressed in several neoplastic tissues. However, the effect of SENP1 in acute promyelocytic leukemia (APL) has not been elucidated. In the present study, it was observed that SENP1 deficiency had no effect on the spontaneous apoptosis or differentiation of NB4 cells. Arsenic trioxide (As2O3) could induce the upregulation of endoplasmic reticulum (ER) stress, resulting in the apoptosis of NB4 cells. Additionally, knockdown of SENP1 significantly increased As2O3-induced apoptosis in NB4 cells transfected with small interfering RNA targeting SENP1. SENP1 deficiency also increased the accumulation of SUMOylated X-box binding protein 1 (XBP1), which was accompanied by the downregulation of the messenger RNA expression and transcriptional activity of the XBP1 target genes endoplasmic reticulum-localized DnaJ 4 and Sec61a, which were involved in ER stress and closely linked to the apoptosis of NB4 cells. Taken together, these results revealed that the specific de-SUMOylation activity of SENP1 for XBP1 was involved in the ER stress-mediated apoptosis caused by As2O3 treatment in NB4 cells, thus providing insight into potential therapeutic targets for APL treatment via manipulating XBP1 signaling during ER stress by targeting SENP1.