Analysis of low molecular weight or metabolic compounds can provide deeper insights into the biochemical pathways regulating normal cell function. Here we report, for the first time, a method to extract a large number of metabolites from rat spermatozoa, which were analysed using gas chromatography mass spectrometry. Based on the retention time index and at least 3-5 fragment qualifier ions, we positively identified 71 compounds, 2 of which we could not find any match to the database. Several different classes of metabolic compounds including amino acids, sugars, fatty acids, sterols and lipids were found. In order to gain insight into sperm function, we extracted metabolites from sperm cells that were in the initial stages of the post-testicular sperm maturation process known as capacitation, and compared the relative intensity of each compound to non-capacitated spermatozoa through the use of an internal standard. We could clearly demonstrate significant down regulation of cholesterol, a hallmark of capacitating cells, being less abundant in the more mature cells. In addition, several monosaccharides including glucose, fructose, sorbitol, galactose and the polyol myo-Inositol decreased in their abundance as sperm begin to capacitate. Interestingly, galactose was able to support sperm motility and an increase in the level of tyrosine phosphorylation, however this came at the expense of longevity of these cells when compared to glucose.