Excessive consumption of sugar may serve as a primary and independent factor for increased morbidity of diabetes. We test the hypothesis that the transient receptor potential vanilloid type 1 (TRPV1) channels play a protective role against sugar-induced pancreatic endoplasmic reticulum (ER) stress, inflammatory cell infiltration, and tissue fibrosis. Wild-type (WT, C57BL/6) or gene-targeted TRPV1-null mutant (TRPV1-/-) mice were fed a normal (CON) or high sucrose diet for 22-23 weeks. TRPV1 ablation impaired glucose tolerance and suppressed glucose-induced insulin release when fed a CON diet (p<0.05). Sucrose intake further impaired glucose tolerance and insulin release in TRPV1-/- compared to WT mice (p<0.05). Sucrose intake increased pancreatic ER stress, showing elevated levels of chop protein content in both strains and GRP78, XBP-1, and CREB-2 in TRPV1-/- mice only (p<0.05). TRPV1 ablation increased levels of phosphorylated JNK (WT, 0.12±0.14; TRPV1-/-, 0.31±0.13) and decreased glut2 contents (WT, 1.38±0.24; TRPV1-/-, 0.83±0.09) in the pancreas when fed a CON diet (p<0.05). Sucrose intake further increased levels of pancreatic phosphorylated JNK (WT, 0.89±0.19; TRPV1-/-, 1.05±0.19) and decreased glut2 contents (WT, 0.39±0.04; TRPV1-/-, 0.26±0.16) in both strains (p<0.05). Sucrose intake increased pancreatic TGF-β levels in both strains with exacerbation in TRPV1-/- mice (p<0.05), and increased pancreatic phosphorylated Smad2/3 and collagen levels in TRPV1-/- mice only (p<0.05). Quantitative immunostaining revealed that sucrose intake enhanced pancreatic infiltration of CD3 positive cells in both strains and macrophage in TRPV1-/- mice only (p<0.05). Thus, TRPV1 ablation impairs insulin release and glucose tolerance at baseline possibly via increased pancreatic phosphorylated JNK signaling and decreased glut2 expression. Sucrose intake aggravates impairment in insulin release and glucose tolerance when TRPV1 is ablation, possibly via enhancing ER stress, macrophage infiltration, and activation of the TGF-β/Smad pathway leading to pancreatic fibrosis, suggesting preserving TRPV1 expression and function may attenuate pancreatic injury via alleviating inflammatory responses.