Helix B surface peptide (HBSP) is an erythropoietin (EPO)-derived peptide that protects tissue from the risks of elevated blood pressure and thrombosis. This study focused on the protection of HBSP in hepatic ischaemia/reperfusion (I/R) by enhancing the level of autophagy. In detail, we randomly divided C57BL/6 mice into sham-operated, hepatic ischaemia/reperfusion (I/R), I/R + HBSP, I/R + HBSP + 3-methyladenine (autophagy inhibitor), I/R + HBSP + rapamycin (mTOR inhibitor), and I/R + HBSP + Ly294002 (Akt inhibitor) groups. We assessed alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) levels in mouse sera, and performed haematoxylin/eosin (HE) staining, immunohistochemistry, electron microscopy, immunofluorescence microscopy, and western blotting on liver tissue to detect the degree of liver injury, liver apoptosis, autophagy, and the expression of microtubule associated protein 1 light chain 3 alpha (Map1lc3, or LC3), Beclin 1, phospho-mTOR, mTOR, phospho-Akt (P-Akt), and Akt. HBSP relieved hepatic I/R injury in a concentration-independent manner. The expression of LC3II, LC3I, and Beclin 1, and the formation of autophagosomes, in the I/R + HBSP group were higher than those in the I/R group. The protective effects of HBSP were abolished by 3-methyladenine and, to a lesser extent, Ly294002, but enhanced by rapamycin. Furthermore, In vivo, HBSP also protected against hypoxia injury induced by cobalt chloride (CoCl2) through improving the level of autophagy. Therefore, HBSP protected against hepatic I/R injury, mainly via regulating autophagy by targeting mTOR.