Sperm are amongst the most rapidly evolving cell types due, in part, to their central role in post-mating competitive reproductive success (sperm competition) and participation in coevolving male-female interactions. To establish the molecular basis of sperm divergence we have conducted a whole-cell, semi-quantitative proteomic approach to characterize sperm divergence in three closely related Mus species (musculus, spretus and spicilegus), which experience different regimes of sperm competition and exhibit remarkable variation in sperm production, energetics, motility and fertilization capacity.