Abstract: Sangguayin preparation (SGY-P) is refined from the traditional Chinese medicinal compound Sangguayin, which “clears heat and promotes fluid” and “tonifies kidney and spleen” for “Xiaoke”, commonly known as ‘Diabetes mellitus’ in clinics. Previous studies have shown that SGY-P could reduce insulin resistance and repair damaged pancreas in db/db mice, but the underlying mechanisms were unclear. Here, we investigated whether treatment with SGY-P could protect pancreatic β-cells from apoptosis and uncovered the underlying mechanisms. db/db mice were used to observe the hypoglycemic and islet protective effect in vivo. Apoptosis was induced in mouse insulinoma 6 (MIN6) cells by palmitate, following which the cells were treated with SGY-P for elucidating the anti-apoptotic mechanism in vitro. Cell viability and nuclear morphology were detected by CCK-8 assay and Hoechst 33258 staining. The expression levels of apoptosis-, endoplasmic reticulum (ER) stress-, and autophagy-related proteins were measured by western blot. The results showed that SGY-P reduced fasting blood glucose, pancreatic pathological changes, and islet β-cell apoptosis in db/db mice. Palmitate-induced apoptosis in MIN6 cells was decreased by SGY-P treatment. Hence, SGY-P therapy exhibited a protective effect on pancreatic β-cells by decreasing the expression of cleaved caspase-3, cleaved PARP and Bax, and increasing Bcl-2 by suppressing ER stress (Bip/XBP1/IRE1α/CHOP/Caspase-12) and autophagy (LC3/p62/Atg5) pathways.2/Atg5) pathways.