AbstractHexadimethrine bromide (Polybrene) was once used clinically as a heparin neutralizer and has recently found use as a promoter in virus-mediated gene therapy trials and gene transfer in research. However, the potential for tissue-specific toxicity of polybrene at low doses has been ignored so far. Here, we found that after intracerebroventricular (ICV) polybrene injection, mice showed disability of movement accompanied neural death and gliosis in brain, and in human neurons, polybrene induces concentration-dependent neuritic beading and fragmentation. Mechanistically, polybrene induces a rapid voltage-dependent calcium channel (VDCC)-mediated influx of extracellular Ca²⁺. The elevated cytoplasmic Ca²⁺ activates DRP1, which leads to mitochondrial fragmentation and metabolic dysfunction. At the same time, Ca²⁺ influx induces endoplasmic reticulum (ER) fragmentation and tightened associations between ER and mitochondria, which makes mitochondria prone to Ca²⁺ overloading and ensuing permeability transition. These results reveal an unexpected neuronal toxicity of polybrene, wherein Ca²⁺ influx serves as a regulator for both mitochondrial dynamics and ER–mitochondrial remodeling.