Pancreatitis is an inflammatory disease of the exocrine pancreas, characterised by auto-digestion of the pancreas and necrotic cell death. Oxidative stress and impairment of intracellular calcium ([Ca²⁺]_i) homeostasis has been implicated in this disease. Previous studies have shown that oxidative stress impairs hormone-evoked Ca²⁺ signalling and induces an irreversible increase in [Ca²⁺]_i (Ca²⁺ overload) (Bruce et al, 2007). This oxidant-induced Ca²⁺ overload response coincided with inhibition of the PMCA and mitochondrial depolarisation. Moreover, this oxidant-induced PMCA inhibition could occur without impairment of mitochondrial Ca²⁺ handling or ATP depletion and was attenuated by inhibitors of the mitochondrial permeability transition pore (Baggaley et al, 2008). Several studies have demonstrated that insulin can activate pro-survival pathways and protect from pancreatic cell injury. Therefore the aim of the current study was to test the effects of insulin on oxidant-mediated impairment of Ca²⁺ homeostasis and inhibition of the PMCA. Pancreatic acinar cells were isolated from Sprague-Dawley rats by collagenase digestion. The effect of hydrogen peroxide (H₂O₂) on resting [Ca²⁺]_i was tested on fura-2-loaded cells treated with or without insulin (1-100nM). In addition, we utilised an in situ Ca²⁺ clearance assay in which the PMCA activity was pharmacologically isolated. A paired experimental design was used to directly compare clearance phases in which H₂O₂ was applied during the second clearance phase and the rate normalised to the first clearance phase. All data are presented as mean values ± standard error. Insulin pre-treatment (1nM) had no effect on the H₂O₂ induced increase in resting [Ca²⁺]_i (50µM: 0.47 ± 0.14 ratio units, n = 3 control and 0.40 ± 0.15 ratio units, n = 4 insulin treated). However, 1nM insulin caused apparent shift in the proportion of cells that recovered following H₂O₂ treatment (100µM: 36% of total cells, n = 5 control and 56% of total cells, n = 6 insulin treated) and an increase in the magnitude of recovery from the H₂O₂-mediated Ca²⁺ overload response (50µM: 27.13 ± 0.93%, n = 3 control and 57.21 ± 9.13%, n = 4 insulin treated). Furthermore, H₂O₂ inhibited the PMCA in a concentration-dependent manner (500µM: rate = 18.44 ± 4.75%, n = 6 compared to control: rate = 91.70 ± 12.54%, n = 8). This inhibition was attenuated in cells treated with 1nM insulin (500µM: rate = 44.53 ± 9.13%, n = 4). In summary, these data suggest that insulin could protect against oxidant-induced Ca²⁺ overload and oxidant-induced inhibition of the PMCA. This may have important implications for the prevention of necrotic cell death associated with pancreatitis. Future work will involve elucidating the molecular mechanisms of this insulin-mediated protection of the oxidant-induced inhibition of the PMCA.