Endoplasmic reticulum stress (ERS) plays an important role in diabetes mellitus (DM), but the association between DM and ERS is unknown. We have previously shown that streptozotocin (STZ)-induced diabetes in rats is characterized by increased levels of ERS markers. Here, we tested whether the chemical chaperone 4-phenylbutyric acid (4-PBA) ameliorated ERS-associated apoptosis in pancreatic β-cells in rats with STZ-induced diabetes. Male Sprague-Dawley rats were divided into 3 groups: control group, DM group, and DM model plus 4-PBA treatment group (4-PBA group). DM model rats were induced by injection of STZ (60 mg/kg) intraperitoneally, and 4-PBA was administered daily by gavage at a dose of 500 mg/kg body weight for 20 days. β-cell apoptosis was higher in the DM group than in the control group. Moreover, the expression of caspase-3, Bax, and the ERS indicators Bip and CHOP was markedly elevated in the pancreas of rats in the DM group, whereas the expression of Bcl-2 was lower in these rats (P < 0.05). Blood glucose concentration in diabetic rats gradually decreased with 4-PBA treatment but remained higher at the end of the experiment compared to the concentration in control rats. Consistent with this, 4-PBA raised the fasting insulin level in diabetic rats; it also suppressed the expression of caspase-3, Bax, and ERS indicators but enhanced the expression of Bcl-2. In conclusion, 4-PBA protects pancreatic β-cells from apoptosis in STZ-induced diabetes by attenuating the severity of ERS.