In vivo matured oocytes collected from dominant, pre-ovulatory follicles have superior developmental competence when compared to their in vitro matured counterparts, indicating that in vitro maturation (IVM) methods are sub-optimal. Cumulus cells (CC) have a critical role during oocyte maturation; thus, their gene expression holds key information regarding cellular processes associated with oocyte development. Understanding these biological events occurring during in vivo maturation of equine oocytes has the potential to improve IVM conditions. The aim of this study was to compare gene expression profiles of CC obtained from in vitro matured oocytes (IVM-CC) and in vivo matured oocytes (IVV-CC) by identifying differentially expressed genes (DEG) and significant biological pathways related to maturation. For the IVM-CC group, cumulus-oocyte complexes (COCs; n =12) were collected and matured in vitro as previously described (Meyers et al. Reprod Fertil Dev. 2019; 12:1874-1884). COCs for the IVV-CC group (n = 12) were collected after gonadotropin induction of ovulation as previously described (Foss et al. EVJ. 2013; 45:39-43). Extracted RNA from CC was sequenced, generating an average of 24 million reads/sample. The raw reads were trimmed (Trimmgalore) and mapped (STAR) to the current equine reference genome. Mapped reads were quantified (Featurecount) and compared between IVM-CC and IVV-CC groups,using the DESeq2 package in R software. A total of 14,479 genes were expressed in the cumulus cells. Principal component analysis showed a uniform pattern of gene expression among the IVV-CC, in contrast to a more scattered pattern in IVM-CC samples. We found 6,677 DEGs with 4,490 downregulated and 2,187 upregulated genes in the IVM-CC group. The enrichment pathway analysis demonstrated that downregulated genes were involved in transmembrane signaling receptors and chemical stimulus detection, while upregulated genes were associated with chromosome segregation, DNA repair, sister chromatid segregation, DNA replication, mitotic cell cycle process, and nuclear division. We found abnormal expression of genes involved in cumulus expansion and gonadotropin receptors in the IVM-CC group. Our data highlights that IVM culture conditions seem to be a distant replica of the in vivo conditions, as demonstrated by the divergent gene expression of the cumulus cells. The difference observed in gene expression and associated pathways could explain the variability in developmental competence between the in vivo and in vitro matured gametes. Lastly, this data serves as a starting point to determine modifications of current in vitro maturation culture protocols necessary to mimic in vivo conditions. This study was funded by the Theriogenology Foundation and Center for Equine Health at UC Davis.