Chitosan is non-toxic, biodegradable and biocompatible. However, it is insoluble in water, which limits its applications in biomedical areas. Hydroxypropyltrimethyl ammonium chloride chitosan (HACC), a chitosan derivative, can be dissolved in physiological condition and has been widely used in the field of biomedicine and bioengineering. The biological effect of HACC has been extensively studied. However, it is rarely investigated at the subcellular level. To study the biological effect of HACC, mitochondria, energy-producing organelles in eukaryotes, were chosen as a model. The investigation mainly focused on the changes of mitochondrial membrane property in the presence of HACC. Results showed that HACC can induce the collapse of mitochondrial transmembrane potential (∆Ψm), the increase in mitochondrial membrane swelling and the decrease of mitochondrial membrane fluidity, demonstrating that mitochondrial membrane permeability transition pore (mPTP) opening happened. Possible mechanism of mPTP opening investigation indicated that it was occurred in a typical model. In addition, HACC can induce the release of cytochrome C (Cyt c) and affect the respiratory activity of mitochondria. The study will provide a lot of important information on biosafety evaluation of HACC.