Apoptosis is a major mode of cell death occurring during ischemia-reperfusion (I/R) induced injury. The p66(Shc) adaptor protein, which is mediated by PKCβ, has an essential role in apoptosis under oxidative stress. This study aimed to investigate the role of PKCβ2/p66(Shc) pathway in intestinal I/R injury. In vivo, ischemia was induced by superior mesenteric artery occlusion in mice. Ruboxistaurin (PKCβ inhibitor) or normal saline was administered before ischemia. Then blood and gut tissues were collected after reperfusion for various measurements. In vitro, Caco-2 cells were challenged with hypoxia-reoxygenation (H/R) to simulate intestinal I/R. Translocation and activation of PKCβ2 were markedly induced in the I/R intestine. Ruboxistaurin significantly attenuated gut damage and decreased the serum levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). Pharmacological blockade of PKCβ2 suppressed p66(Shc) overexpression and phosphorylation in the I/R intestine. Gene knockdown of PKCβ2 via small interfering RNA (siRNA) inhibited H/R-induced p66(Shc) overexpression and phosphorylation in Caco-2 cells. Phorbol 12-myristate 13-acetate (PMA), which stimulates PKCs, induced p66(Shc) phosphorylation and this was inhibited by ruboxistaurin and PKCβ2 siRNA. Ruboxistaurin attenuated gut oxidative stress after I/R by suppressing the decreased expression of manganese superoxide dismutase (MnSOD), the exhaustion of the glutathione (GSH) system, and the overproduction of malondialdehyde (MDA). As a consequence, ruboxistaurin inhibited intestinal mucosa apoptosis after I/R. Therefore, PKCβ2 inhibition protects mice from gut I/R injury by suppressing the adaptor p66(Shc)-mediated oxidative stress and subsequent apoptosis. This may represent a novel therapeutic approach for the prevention of intestinal I/R injury.