This study aimed to evaluate gene expression for embryos collected 8±1.5 days after ovulation, of different sizes. Non-lactating Saddlebred mares located on two experimental farms were inseminated with semen from one stallion per farm after hCG induction. Mares were examined for ovulation at 24 or 48 h after hCG, depending on the farm. Twenty-eight embryos (11 and 17, respectively) were collected 8 days after ovulation was confirmed, with each embryo from a different dam. Embryos were measured and bisected to obtain trophoblast (TE) or inner cell mass enriched trophoblast (TE-ICM). Paired end, non-oriented RNA sequencing was performed (Illumina, NextSeq500) on both samples of embryos of different diameters: Small (<700µm, mean of 560±85µm, n=9), Medium (700-1200µm, 886±160µm, n=11) and Large (>1,200µm, 1,719±488µm, n=8). For TE-ICM data, deconvolution (DeMixT) was used to discriminate gene expression in ICM vs TE. Differential expression was analyzed (DESeq2) with farm and embryo sex as cofactors (false discovery rate (FDR) <0.05 cutoff). Overrepresentation tests were performed on differentially expressed genes (DEG) using PANTHER software with GObp database. Within the 14,249 and 13,406 genes expressed in ICM and TE, respectively, 642 in ICM and 123 DEG in TE were observed in Small vs Medium while 1,228 in ICM and 443 DEG in TE were identified in Large vs Medium. Of particular interest, in the ICM and TE, when compared to Medium embryos, Insulin Like Growth Factor(IGF)2 was more expressed in Small (log2 Fold Change, log2FC=1.9 and 2.0 for ICM and TE, respectively, FDR<0.05) while IGF1 was up-regulated in ICM (log2FC=2.0, FDR<0.05) and down-regulated in TE (log2FC=-1.2, FDR<0.05) in Large embryos. IGF1 is known to stimulate estrogen production in pig embryos. Here, several P450 cytochromes including cytochrome P450 aromatase (CYP19A1, log2FC=4.46, FDR<0.0001) associated with 17β-Hydroxysteroid dehydrogenase (HSD17B1, log2FC=0.58, FDR<0.05) were upregulated in the ICM in Large vs Medium. Moreover, Nanog Homeobox gene (log2FC=-3.0, FDR<0.0001) and several SRY-Box Transcription Factors (SOX), including SOX2 (log2FC =-2.3, FDR<0.01) were downregulated in the ICM of Large vs Medium. In ICM, upregulated DEGs in Small vs Medium were principally involved in cell proliferation and downregulated DEGs in mitochondria and regulation of apoptosis while in upregulated DEGs Large vs Medium, steroid biosynthesis, cell growth and migration and lipid transport were overrepresented. In TE, no gene set was overrepresented in Small vs Medium, while downregulated DEGs in Large vs Medium were involved in DNA methylation and amino acid metabolism. In conclusion, equine embryos of different diameter differ in gene expression associated with developmental parameters. Differences in gene expression between ICM and TE suggest increased secretory activity in ICM with embryo growth.