Acrylamide (AA), a well-known neurotoxin, shows obvious damage to the nervous system. Cyanidin-3-O-glucoside (C3G), a representative anthocyanin, is identified as a promising neuroprotective agent as its excellent antioxidant capacity. This study evaluated the mitoprotective effects of C3G against AA-mediated neurotoxicity. The results showed that pretreatment with C3G (10 μmol/L) significantly lessened the reduction in AA-induced cell survival rate, increasing cell viability by 1.31 times compared to the AA-only group. C3G reduced intracellular ROS and MDA level accumulation by 84.0% and 61.9%, respectively. Furthermore, C3G suppressed the activation of NLRP3 inflammasome and Caspase-3-dependent apoptosis pathways induced by AA. Further mitochondrial analysis revealed that C3G pretreatment enhanced mitochondrial membrane potential recovery by 1.50 times and preserved the mitochondrial ultrastructure, while also restoring the aerobic respiratory capacity. PCR array demonstrated that C3G reversed the AA-induced downregulation of mitochondrial biogenesis genes PGC-1α and TFAM by 2.67-fold and 1.88-fold, respectively, and mitochondrial dynamics genes Mfn2 and Opa1 by 2.76-fold and 3.08-fold. Further in vivo studies confirmed that the blueberry anthocyanin extracts, which are mainly composed of C3G, showed neuroprotective function through maintaining mitochondrial function, alleviating inflammation, and apoptosis. This article provides new insights into the neuroprotective effects of C3G.