Introduction: The aim of this study was to investigate whether Cortexin®, a brain peptide-containing agent, has any mitigating effect on high glucose-induced neuropathy, using primary cultured rat sensory neurons. Materials and Methods: Dorsal root ganglia (DRG) were excised from decapitated adult rats. Individual neurons were isolated following enzymatic and mechanical procedures. Cells were seeded on E-plate® with gold microelectrodes and maintained in conventional culture media in a CO2 incubator at 37°C. After allowing for 24 h for cell adhesion and recovery from acute enzymatic trauma, neurons were exposed to high glucose (HpG) in the absence and presence of different concentrations of Cortexin® (2–40 µg/mL). Neuroprotective effects were followed with the Real-Time Cell Analyzer® by utilizing measurement of Cell Index, a parameter representing cell viability, cell attachment, and neurite outgrowth. Results: Exposure of DRGs to HpG (50 m<sc>m</sc>) caused a rapid and sustained decrease in the mean area under the curve (AUC, values derived from time vs. Cell Index curve) compared to the mean AUC values in normoglycemic (NG) wells. Co-treatment with Cortexin® attenuated this HpG-induced effect, in a concentration-dependent manner (NG: 1.00 ± 0.00 vs. HG: 0.18 ± 0.02, p < 0.05; and HpG + Cortexin® [40 µg/mL]: 0.66 ± 0.17, p = 0.002 versus HpG). In normoglycemic conditions, Cortexin® treatment led to a concentration-dependent increase in the mean AUC values. Conclusions: Data from this in vitro study suggest that Cortexin® has potential neuroprotective effects against chronic hyperglycemic insult in rat sensory neurons. Our results warrant further in vivo studies and may have clinical implications for diabetes-associated peripheral neuropathy.