Platelet-activating factor (PAF) is a well-described autocrine growth factor involved in several reproductive processes and is tightly regulated by its hydrolysing enzyme, PAF acetylhydrolase 1B (PAFAH1B). This intracellular enzyme consists of three subunits: one regulatory, 1B1, and two catalytic, 1B2 and 1B3. PAFAH1B3 has remained uncharacterised until now. Here, we report that PAFAH1B3 is present during the different stages of the first meiotic division in bovine, murine and human oocytes. In these species, the PAFAH1B3 subunit was clearly present in the germinal vesicle, while at metaphase I and II, it localised primarily at the meiotic spindle structure. In cattle, manipulation of the microtubules of the spindle by nocodazole, taxol or cryopreservation revealed a close association with PAFAH1B3. On the other hand, disruption of the enzyme activity either by P11, a selective inhibitor of PAFAH1B3, or by PAFAH1B3 antibody microinjection, caused arrest at the MI stage with defective spindle morphology and consequent failure of first polar body extrusion. In conclusion, our results show that one of the catalytic subunits of PAFAH1B, namely PAFAH1B3, is present in bovine, murine and human oocytes and that it plays a functional role in spindle formation and meiotic progression during bovine oocyte maturation.