For the ex-situ conservation of giant pandas, both collecting and preserving semen are important methods. The seminal plasma is rich in nutrients and bioactive substances, such as proteins, carbohydrates, lipids, amino acids, and hormones, which play an important role in the reproduction and reproductive health of the species. This is the first study to analyze the seminal plasma proteins of giant pandas through proteomics and identified 1125 proteins. These proteins are related to protein turnover, translation, and metabolism. The seminal plasma proteins of giant pandas were then compared to those of humans, pigs and sheep, with many unique proteins found in giant panda samples. Among these proteins, the WD40 repeat-containing proteins have been identified and implicated in sperm function and fertility. Understanding the composition and function of proteins in the giant panda seminal plasma proteome can provide valuable insights into their reproductive biology and help develop strategies to improve their reproductive success in captivity, which is essential for giant panda conservation.