Di-(2-ethylhexyl) phthalate (DEHP) and dibutyl phthalate (DBP) are representative phthalic acid esters (PAEs), a class of environmental endocrine disruptors used as plasticizers. PAEs exposure is associated with glucose metabolism, insulin resistance, and glucose tolerance; however, the mechanism and various PAE effects on human glucose metabolism remain largely unknown. In this study, we investigated the effects of DEHP, DBP, and their mixture on rat insulinoma (INS-1) cell apoptosis and the mechanism involved in vitro. The INS-1 cells were cultured in RPMI-1640 + 10% fetal bovine serum for 24 h and pretreated with dimethyl sulfoxide (vehicle, <0.1%), DEHP (30 μM), DBP (30 μM), and their mixture (30 μM DEHP + 30 μM DBP). The methyl-thiazolyl tetrazolium bromide test was used to measure cell viability. Hoechst 33342/propidium iodide (PI) staining and Annexin V-FITC/PI staining, 2′,7′-dichlorofluorescein diacetate assay, and glucose-induced insulin secretion assay were used to detect cell apoptosis rates, intracellular reactive oxygen species (ROS), and insulin secretion in INS-1, respectively. The mRNA expression levels of Bcl-2, Bax, Caspase 9, Caspase 8, Caspase 3, phosphoinositide 3-kinase (PI3K), and Akt were detected using real-time quantitative reverse transcription PCR; their protein expression levels were detected using western blotting. To the best of our knowledge, this study was the first to show that the combined effect of the two PAEs promotes a ROS-mediated PI3K/Akt/Bcl-2 pathway-induced pancreatic β cell apoptosis that is significantly higher than the effects of each PAE. Thus, safety standards and studies do not consider this effect as a significant oversight when blending PAEs. We assert that this must be addressed and corrected for establishing more impactful and safer standards. Schematic diagram of the phosphoinositide 3-kinase (PI3K)/Akt/Bcl-2 signaling pathway involved in β-cell dysfunction induced by DEHP, DBP, and their mixture. Our results show that exposure to DEHP and/or DBP can induce oxidative damage and apoptosis in INS-1 cells and decrease insulin secretion. The primary underlying mechanisms include inhibition of the PI3K/Akt/Bcl-2 signaling pathway and enhanced expression of cleaved-Caspase 9, cleaved-Caspase 3, and Bax in INS-1 cells through oxidative damage. Display omitted •INS-1 cell proliferation was decreased by DEHP, DBP, and their mixture.•ROS production in the DEHP, DBP, and mixture groups was higher than that in the control.•PI3K, p-Akt, and Bcl-2 mRNA expression in DEHP, DBP, and mixture group was decreased.