Following ejaculation, mammalian spermatozoa undergo an obligatory process known as capacitation, which enables these cells to bind to and fertilize an oocyte. Since spermatozoa are transcriptionally and translationally silent, the functional metamorphosis of these cells during capacitation is accomplished entirely by PTMs. Despite the importance of this process, very few studies have attempted to define the precise nature of the proteomic changes that allow spermatozoa to attain a capacitated state. Here we report the use of an IPG-strip pre-fractionation approach to isolate and purify tryptic peptides derived from mouse spermatozoa exhibiting varying degrees of capacitation. Following focusing, the strips were cut into 1 cm segments, the peptides extracted and run into a mass spectrometer. Label-free, quantitative analysis of proteomic changes associated with capacitation was then performed. In total, we found 210 significant peptide changes. Of these, we could conclusively interpret the tandem mass spectra of 71 peptides, corresponding to 52 protein changes during capacitation. Many proteins including VDAC2, Fascin-3 and sorbitol dehydrogenase (SORD) have not previously been implicated in this process. To validate our data, we were able to show significant upregulation of SORD activity during capacitation, suggesting that the polyol pathway is activated during this process.