Objective: To compare urine proteome changes in the first two days after mating behavior in male rats.

Methods: The urine samples of Sprague-Dawley rats on the day of mating and the day after mating were collected and analyzed by non-label quantitative proteomics by high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS), and the differential proteins in the urine proteome were screened protein function and biological pathways were analyzed.

Results: 43 differential proteins were identified by comparing the urine proteome of rats on the next day of mating with that on the day of mating, and nearly two-thirds of the differential proteins were related to spermatogenesis.

Conclusions: There were changes in the urinary proteome on the next day of mating compared with the day of mating, and some of the proteins whose functions were known to be related to spermatogenesis. Urine proteome has the potential to reflect spermatogenesis.