In mammalian sperm, mitochondria are very densely packed and form a helical sheath in the midpiece of the flagellum. Mitochondria from somatic cells can rapidly change shape to adapt to environmental conditions. During capacitation, mammalian spermatozoa undergo morphological and physiological changes to acquire fertilization ability, evidenced by changes in sperm motility patterns (hyperactivation) and the ability to perform the acrosome reaction. Whether there are changes in sperm mitochondrial morphology during capacitation is unknown. This work aimed to quantify morphometric changes in the sperm midpiece during capacitation. Using mitochondrial fluorescent probes and a combination of freely available software, we quantified the dimensions and fluorescence intensity of the midpiece. After capacitation, the area occupied by the mitochondria decreased due to a reduction in the width but not the length of the midpiece. The decrease in the area of the midpiece occurred in spermatozoa that underwent the acrosome reaction, suggesting a reorganization of the mitochondria during capacitation. Ultrastructural analysis supported these results. The application of image processing to fluorescence microscopy images may help to identify morphological changes during capacitation.