Endoplasmic reticulum (ER) stress and subsequent apoptosis play a vital role in myocardial ischemia reperfusion (IR) injury. Fatty acid binding protein 4 (FABP4) may induce ER stress. The aim of this study was to investigate the mechanism and effect of FABP4 on IR injury in vitro. Rat H9c2 cells were exposed to hypoxia reoxygenation (HR) to create an IR model in vitro. FABP4 was overexpressed in HR-injured H9c2 cells. Transfection with FABP4 siRNA increased cell viability and decreased LDH upon HR stimulation. FABP4 cessation also suppressed apoptotic cells and caspase-3 activity after HR. Downregulation of FABP4 significantly inhibited ER stress by decreasing the protein expression of p-PERK, GRP78, and ATF6. FABP4 silencing also restrained the ER stress-mediated apoptotic pathway, as indicated by decreased pro-apoptotic proteins p-JNK, CHOP, Bax, and caspase-12, as well as upregulation of Bcl-2 during HR. Furthermore, FABP4 silencing activated the PI3K/Akt pathway. Blocking this pathway by the specific PI3K inhibitor-LY294002 restored HR-induced ER stress and subsequently reversed the protective effect of FABP4 silencing on HR injury. Taken together, our findings revealed that FABP4 silencing exerts protective effects against HR injury in H9c2 cells through inhibiting ER stress-induced cell apoptosis via activation of the PI3K/Akt pathway.