Abstract: Although elements of type I and II apoptosis have been demonstrated in human spermatozoa, their functionality has not been evaluated. The first dynamic studies revealed no type I apoptosis signal transduction via CD95. The aim of our study was to clarify whether type II apoptosis can be induced in human spermatozoa. Betulinic acid, a cytotoxic agent and highly specific inductor of type II apoptosis, acts by a direct effect on mitochondria. Motility, mitochondrial transmembrane potential, and active caspase-9 and -3 were examined in human ejaculated spermatozoa of 33 semen samples from healthy volunteers after incubation with 60 μg/mL betulinic acid for 10 and 60 min, respectively. Untreated aliquots of each sample served as negative controls. Treatment with betulinic acid resulted in the induction of type II apoptosis measured by disruption of mitochondrial transmembrane potential and activation of caspase-9 and -3. The loss of mitochondrial energy supply resulted in a significant decrease of spermatozoal motility and velocity. In spermatozoa, mitochondria are tightly packed and located exclusively in the midpiece region. This might contribute to their susceptibility against the induction of type II apoptosis and should be considered for therapeutic interventions and might have a future in the development of advanced birth control methods.