Polycomb-repressive complex 2 (PRC2) catalyzes the methylation of histone H3 Lys27 (H3K27) and functions as a critical epigenetic regulator of both stem cell pluripotency and somatic differentiation. EZH1 and EZH2 are enzymatic subunits of PRC2, the latter of which is encoded by alternative splice isoforms. To elucidate how EZH variants regulate PRC2 in establishment of H3K27 methylaiton during cell differentiation, we analyzed the alteration of di- versus tri-methylation of H3K27 in spermatocytes during meiotic prophase I. We found that H3K27me2 levels were strikingly elevated across all nuclei during the zygotene-to-pachytene stage transition, which occurs along with upregulation of expression of Ezh2. In contrast, there was no significant increase in H3K27me3 during meiotic progression. Interestingly, a new Ezh2 isoform, where exon 14 is deleted due to alternative splicing, was dramatically increased in meiocytes compared to mitotic germ cells. However, the Ezh2 isoform containing exon 14 (Ezh2- ex14) downreguated as meiosis was initiated. We observed that expression of Ezh2-ex14 isoform upregulated during mitosis in rapidly dividing mouse embryonic fibroblasts. These results suggest that EZH2 isoforms may function differently in non-dividing cells such as spermatocytes undergoing differentiation during meiosis versus mitotically replicating cells such as mouse embryonic fibroblasts and that these distinct isoforms may determine the choice of di- versus tri-methylation of H3K27.