Objective: To observe the effect of PI3Kγ gene knockout on the extent of the pancreatic lesion, acinar cell function and HSP70 protein expression level of mice with acute pancreatitis (AP). Methods: The male wild-type (WT) C57BL/6 mice and PI3Kγ gene knockout (KO) mice were randomly divided into control group and AP group with 12 mice in each group. The in vivo AP model was induced by intraperitoneal injections of cerulean and mice of the control group were subjected to exactly the same regimen of saline injections. In addition, the pancreatic acini were isolated from another two different sets of mice (8 per set), and then the acinar cells were stimulated with CCK-8 to prepare an in vitro AP model. Control cells were treated with DMSO instead of CCK-8. Pathological changes of the pancreatic tissues were assessed, and the serum level of amylase, trypsin activity in pancreatic tissues and acinar cells, and level amylase release from the acinar cells were measured. The HSP70 protein expressions in pancreatic tissues and acinar cells were determined by Western blot analysis. Results: In pathological observation, the pancreatic tissues from the control groups of both types of mice showed no abnormality, while both AP groups presented varying degrees of edema, necrosis and hemorrhage. The quantitative analysis showed that the number of necrotic acinar cells and vacuoles of the KO mice were significantly less than those of the WT mice (both P<0.05). There were no significant differences in trypsin activity of the pancreatic tissues and isolated acinar cells between the two control groups (both P>0.05), but the trypsin activity of the pancreatic tissues and isolated acinar cells of the KO mice were significantly lower than those of the WT mice in the AP groups (in vivo and in vitro) (both P<0.05). No statistical differences were noted in serum amylase level and acinar cell amylase release curve between the two types of mice (both P>0.05). Compared with the control groups, the HSP70 protein expressions in both pancreatic tissues and acinar cells increased obviously in AP groups (in vivo and in vitro), in which the HSP70 expression levels of the KO mice were significantly higher than those of the WT mice (both P<0.05). Conclusion: PI3Kγ may promote acinar necrosis in acute pancreatitis by down-regulating HSP70 protein expressions and enhancing activation of trypsinogen, but it has no obvious effect on amylase secretion.