Sperm are exposed to substantially different environments during their life history, such as seminal fluid or the female sexual tract, but remarkably little information is currently available about whether and how much sperm composition and function alters in these different environments. Here, we used the honeybee Apis mellifera and quantified differences in the abundance and activity of sperm proteins sampled either from ejaculates or from the female’s sperm storage organ. We find that stored and ejaculated sperm contain the same set of proteins but that the abundance of specific proteins differed substantially between ejaculated and stored sperm. Most proteins with a significant change in abundance are related to sperm energy metabolism. Enzymatic assays performed for a subset of these proteins indicate that specific protein activities differ between stored and ejaculated sperm and are typically higher in ejaculated compared to stored sperm. We provide evidence that the cellular machinery of sperm is plastic and differs between sperm within the ejaculate and within the female’s storage organ. Future work will be required to test whether these changes are a consequence of active adaptation or sperm senescence and whether they alter sperm performance in different chemical environments or impact on the cost of sperm storage by the female. However, these changes can be expected to influence sperm performance and therefore determine sperm viability or sperm competitiveness for storage or egg fertilization.