Isosteviol (ISV), a diterpene molecule, is an isomer of the backbone structure of a group of substances with proven antidiabetic capabilities. The aim of this study was to investigate if ISV elicits dynamic insulin release from pancreatic islets and concomitantly is able to ameliorate gluco-, lipo-, and aminoacidotoxicity in clonal β-cell line (INS-1E) in relation to cell viability and insulin secretion. Isolated mice islets placed into perifusion chambers were perifused with 3.3 mM and 16.7 mM glucose with/without 10−7 M ISV. INS-1E cells were incubated for 72 h with either 30 mM glucose, 1 mM palmitate or 10 mM leucine with or without 10−7 M ISV. Cell viability was evaluated with a Cytotoxic Fluoro-test and insulin secretion was measured in Krebs-Ringer Buffer at 3.3 mM and 16.7 mM glucose. In the presence of 3.3 mM glucose, 10−7 M ISV did not change basal insulin secretion from perifused islets. However, at a high glucose level of 16.7 mM, 10−7 M ISV elicited a 2.5-fold increase (−ISV: 109.92 ± 18.64 ng/mL vs. +ISV: 280.15 ± 34.97 ng/mL; p < 0.01). After 72 h gluco-, lipo-, or aminoacidotoxicity in INS-1E cells, ISV treatment did not significantly affect cell viability (glucotoxicity, −ISV: 19.23 ± 0.83%, +ISV: 18.41 ± 0.90%; lipotoxicity, −ISV: 70.46 ± 3.15%, +ISV: 65.38 ± 2.81%; aminoacidotoxicity: −ISV: 8.12 ± 0.63%; +ISV: 7.75 ± 0.38%, all nonsignificant). ISV did not improve impaired insulin secretion (glucotoxicity, −ISV: 52.22 ± 2.90 ng/mL, +ISV: 47.24 ± 3.61 ng/mL; lipotoxicity, −ISV: 19.94 ± 4.10 ng/mL, +ISV: 22.12 ± 3.94 ng/mL; aminoacidotoxicity: −ISV: 32.13 ± 1.00 ng/mL; +ISV: 30.61 ± 1.54 ng/mL, all nonsignificant). In conclusion, ISV acutely stimulates insulin secretion at high but not at low glucose concentrations. However, ISV did not counteract cell viability or cell dysfunction during gluco-, lipo-, or aminoacidotoxicity in INS-1E cells.