Objective To explore whether the protective effect of Exendin-4 on lipotoxicity induced by palmitic acid (PA) was mediated by toll-like receptor 4 (TLR4) /c-Jun amino-terminal kinase (JNK)/Bcl-2 associated X protein gene (Bax) pathway in βTC6 cells. Methods The lipotoxic cell model of βTC6 was established and treated with TLR4 inhibitor (CLI-095), JNK inhibitor (SP600125) or Exendin-4. Insulin secretion and protein expression of TLR4, p-JNK, JNK, Bax and Bcl-2 in βTC6 cells were detected. Results (1) Compared with the control group, PA down-regulated glucose-stimulated insulin secretion (GSIS) (P<0.05), up-regulated TLR4 and p-JNK expression level (P<0.05) and down-regulated the Bcl-2/Bax ratio (P<0.05). (2) CLI-095 or SP600125 improved GSIS (P<0.05) and ratio of Bcl-2/Bax against lipotoxicity induced by PA (P<0.05). (3) Exendin-4 also increased GSIS (P<0.05), decreased the expression of TLR4 and p-JNK (P<0.05) and increased ratio of Bcl-2/Bax (P<0.05). Inhibiting the activity of TLR4 or JNK can enhance the effect of Exendin-4 on improving the insulin secretion of β-cells which induced by lipotoxicity, while increasing the activity of TLR4 or JNK antagonizes the effect of Exendin-4 on improving insulin secretion. Conclusions PA may induce the dysfunction of β cell through TLR4/JNK/Bax signal pathway and Exendin-4 could partly protect β cell against PA.