Mitochondria are known to be the main players in important mitochondrial bioenergetic functions such as ATP synthesis, thermoregulatory energy dissipation, Ca2+ transport, generation of reactive oxygen species and mediation of intrinsic apoptosis. Naturally occurring polyamines, due to their high pka are almost completely protonated at physiological pH and behave as polycations in their interactions with mitochondrial membranes. Thanks to these interactions, polyamines are transported electrophoretically into the mitochondrial matrix, where they exhibit a number of effects of significant importance for the above-mentioned mitochondrial functions, particularly inner membrane permeability transition (MPT). This event is closely correlated with the intrinsic occurrence of apoptosis, so that the effect of polyamine interactions with mitochondria has important implications in the pathophysiological consequences of inducing apoptosis, i.e., protection against cancer and neurodegenerative diseases. This review also provides some answers to the old debated problems regarding the possible interactions of polyamines with mitochondrial DNA, overcoming of the Born charging energy by spermine, the ΔΨ threshold value for polyamine transport, and protection of MPT by spermine in in vivo conditions. In conclusion, the old question: “What do polyamines do?” is partially solved.