Inflammatory-related genes are commonly expressed at a low abundance. However, these can still be detected in fecal RNA isolated from dairy calves, which contains a negligible amount of RNA from immune cells under non-diarrheic conditions. Additionally, genes related to common functions of the gastrointestinal (GI) tract were observed in total RNA from fecal samples. However, fecal RNA isolation remains a challenge, because of the potential enrichment of bacterial RNA, which can dilute the targeted eukaryotic RNA and consequently dampen the sensitivity of the fecal RNA method. Therefore, our objective in this study was to determine the differential eukaryotic RNA enrichment in total RNA vs mRNA from same fecal samples of healthy neonatal dairy calves. To test this, 200 mg of feces were used from 6 neonatal Holstein calves for total RNA isolation, using a Trizol based method along with the RNeasy Plus Mini Kit (Qiagen). Then, 45 µg of fecal total RNA was used to isolate mRNA through a magnetic selection using Dynabeads Oligo (dT)25 (Invitrogen). The cDNA synthesis was performed using the SuperScript IV reverse transcriptase (Invitrogen). The standard curve was composite from all samples including cDNA from total RNA and mRNA. The internal control genes used in this experiment were B2M, ACTB, GAPDH, RPS9, and PPIA. Normalized gene expression data were log-transformed before statistical analysis using the Proc Mixed of SAS (SAS 9.4). Expression of genes specific for epithelial cells including cytokeratin 8 (KRT8) and aquaporin (AQP3) as well as inflammatory-related genes (TLR4 and IL1B) were evaluated. The expression of KRT8 was greater (P = 0.03) in fecal mRNA than in fecal total RNA. A trend (P = 0.09) was observed for greater expression of TLR4 in fecal total RNA than in fecal mRNA. The expression of AQP3 and IL1B was not different. Greater expression of KRT8 in mRNA than in total RNA suggests that this additional selection of gene transcripts within fecal RNA might improve the sensitivity of this method and consequently the accuracy and robustness. These results further confirms that the fecal RNA method has a potential to be used as a tool to evaluate GI tract molecular adaptations in dairy calves.