The aim of the present study was to investigate the influence of the protein content of seminal plasma on the motility, viability and acrosome integrity of spermatozoa in extended semen stored for 3 days. A total of 32 semen samples (from four boars) with high (4 mg/ml) and 32 semen samples (from four boars) with low (2 mg/ml) protein content were investigated. The semen samples were diluted by BTS at a ratio of 1:4, and stored for 72 h at 17o C. The percentages of live sperm (LS), live sperm with damaged acrosome (LDA) and total sperm with damaged acrosome (TDA) were detected by flow cytometry. Sperm progressive motility (PM) was detected using CASA. After 72 h of storage, the percentage of LS and PM was significantly (P < 0.01) higher, and the LDA and TDA were significantly (P < 0.01) lower in samples with high protein content than in the samples with low protein content (LS = 66 vs. 44%, PM = 64 vs. 48%, LDA = 15 vs. 21% and TDA = 29 vs. 45%, respectively). When comparing the difference between 0 and 72 h of storage, the percentage decrease in LS and PM, while increase in LDA and TDA were significantly higher in the samples with low (LS: 75 to 44%; PM: 68 to 48%; LDA: 11 to 21% and TDA: 23 to 45%) than in the samples with high protein content (LS: 78 to 66%; PM: 70 to 64%; LDA: 9 to 15% and TDA: 17 to 29%). We concluded that protein content in seminal plasma has a significant influence on progressive motility, viability and acrosome integrity in diluted semen stored for 3 days.