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The buffalo seminal plasma protein profile and its relationship with sperm quality have not been studied in detail. Thus, the aim of the present study was to profile buffalo seminal plasma proteins and to assess the relationship between differentially expressed proteins and sperm characteristics. Semen samples (n = 44) were collected from 11 Murrah buffalo bulls (four ejaculates from each animal) and seminal plasma protein profiling was performed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Matrix-assisted laser desorption ionisation time-of-flight analysis of one of the differentially expressed proteins, namely the 11-12 kDa protein, identified it as tuberoinfundibular peptide of 39 residues (TIP39). Western blot analysis confirmed the presence of TIP39, with TIP39 expression in seminal plasma varying among bulls. Based on TIP39 levels, bulls were classified into two groups, those with high and low protein. The percentages of spermatozoa positive for mitochondrial membrane potential test, chromatin distribution test, synthetic media sperm penetrability test and acrosomal integrity test were significantly (P < 0.05) high in the high protein group. The present study is the first to demonstrate the presence of TIP39 in buffalo seminal plasma and the positive effect of TIP39 on the functional parameters and fertilising ability of spermatozoa.