Trametenolic acid B (TAB) was a lanostane-type triterpenoid isolated from the trametes lactinea (Berk.) Pat. We have previously reported that extract from trametes lactinea (Berk.) Pat and TAB could efficiently improve learning and memory ability of the cerebral ischemia injury rats and suppress mitochondrial-mediated apoptosis in hydrogen peroxide damaged SH-SY5Y cells. However, the potential mechanisms have not been fully understood yet. The current study was to further investigate the protective effect of TAB on oxygen glucose deprivation/reoxygenation (OGD/R)-damaged SH-SY5Y cells and cerebral ischemia/reperfusion (I/R) injury rats, as well as its mechanisms involved. Cell experiments demonstrated that TAB (10, 20 and 40 μg/mL) protected OGD/R-induced SH-SY5Y cell injury by promoting cell proliferation and suppressing LDH leakage; Meanwhile, the results in vivo showed that TAB (20, 40 and 80 mg/kg) might significantly ameliorate the neurological deficit score, cerebral edema, neuronal cell loss and apoptosis, suppress cerebral infarction volume of the cerebral I/R injury rats. Further studies in vitro and in vivo indicated TAB could efficiently reduce OGD/R-damaged SH-SY5Y cell and cerebral I/R rat serum ROS, LDH and MDA levels, elevate SOD, GSH-Px and CAT activities, downregulate miR-10a mRNA and Bax, cytochrome C, cleaved-caspase-3 and cleaved-caspase-9 protein expressions, upregulate p-PIK3CA, p-Akt, p-mTOR, Bcl-2, pro-caspase-9 and pro-caspase-3 protein expressions and p-PIK3CA/PIK3CA, p-Akt/Akt, p-mTOR/mTOR ratios (P < 0.05 or P < 0.01, respectively). Our present study indicated that TAB possessed neuroprotective property against ODG/R and I/R injury by suppressing miR-10a expression, activating PI3K/Akt/mTOR signaling pathway, thereby reducing mitochondrial-mediated apoptosis, which provided a new insight for interpreting the underlying mechanisms of TAB' neuroprotective effect and a candidate agent to treat cerebral I/R injury.