Phospholipase C zeta 1 (PLCζ1), which transcribes a key protein, has an important function in oocyte activation and embryo development because PLCζ1 can trigger a series of intracellular Ca2+ oscillations in mammals. In this study, a novel splice variant in the testis tissues of adult and fetal Chinese Holstein bulls was characterized by reverse transcription-polymerase chain reaction (RT-PCR) and sequencing analysis. The novel splice variant PLCζ1-sv1 was derived from the PLCζ1 complete transcript (PLCζ1-complete) by alternative splicing; the alternative splicing pattern exhibited alternative 5'-splice sites. The full-length transcript, PLCζ1-complete, is the main transcript found in fetal and adult cow testis tissue. Quantitative real-time PCR (qPCR) analysis demonstrated that the expression levels of the PLCζ1-complete transcript were significantly higher than those of the PLCζ1-sv1 splice variant in bovine testis tissues. PLCζ1 protein sequencing analysis showed that the amino acids at positions 453 to 457 were deleted in PLCζ1-sv1, thereby terminating transcription prematurely. In summary, this study provided information to elucidate the structure and function of the bovine PLCζ1 gene.