Objective: Costus afer Ker Gawl is an African ethnomedicinal plant used as a therapeutic for the treatment of oxidative stress-related diseases. This study was designed to identify the most active fractions of C. afer leaves and stem against calcium(II) (Ca2+)-induced mitochondrial membrane permeability transition (MPT). Methods: C. afer leaves and stem were extracted using 70% methanol and subsequently subjected to successive solvent partitioning method to separate it into hexane, ethyl acetate, n -butanol, and aqueous fraction of C. afer leaves and stem. Rat liver mitochondrial fractions were isolated using differential centrifugation technique, and mitochondrial MPT was assessed using a spectrophotometric method. Results: Data showed that n -butanol fractions of C. afer leaves and stem exhibited a significantly high inhibition of Ca2+-induced mitochondrial MPT than other test fractions and standard spermine. Conclusion: n -butanol fractions of C. afer leaves and stem possess bioactive compounds with chemoprotective activity against Ca2+-induced mitochondrial MPT. Furthermore, it is suggested that n -butanol fractions of C. afer leaves and stem could be harnessed for bioactive compounds that can be used in the treatment of mitochondria-associated neurodegenerative diseases.