The ubiquitin-proteasome system plays an important role in many cellular processes through degradation of specific proteins. Low molecular mass polypeptide 2 (LMP-2 or _1i) is one important subunit of the immunoproteasome. Ischemic preconditioning (IPC) activates cell signaling pathways and generates cardioprotection but has not been linked to LMP-2 function previously. LMP-2 knockout mice (C57BL6 background) and wild-type C57BL6 mice were subjected to 30 min of ischemia (I-30) and 120 min of reperfusion (R-120) with or without preceding IPC (10 min of infusion and 5 min of reperfusion). IPC significantly increased left ventricular developed pressure and decreased infarct size in wild-type mice, but this protective effect of IPC was lost in LMP-2 knockout mice. IPC-mediated degradation of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) and activation of the downstream protein kinase Akt were impaired in LMP-2 knockout hearts. The impairment of PTEN degradation was associated with defective immunoproteasomes and decreased proteolytic activities. When LMP-2 knockout mice were pretreated with the PTEN inhibitor bpV(HOpic), cardiac function was significantly improved, and myocardial infarct size was significantly reduced after I-30/R-120. In conclusion, LMP-2 is required for normal proteasomal function and IPC induction in the heart. Its action may be related to PTEN protein degradation.—Cai, Z. P., Shen, Z., Van Kaer, L., Becker, L. C. Ischemic preconditioning-induced cardioprotection is lost in mice with immunoproteasome subunit low molecular mass polypeptide-2 deficiency.