To understand the possible role microRNA (miRNA) have during the onset of fatty liver, we applied an in-silico approach to identify a miRNA signature from a liver transcriptome database. The database was generated from liver tissue of cows developing ketosis and fatty liver during the first 2 wk postpartum. Liver tissue (n = 7/group) for gene expression profiling was sampled at the onset of signs of clinical ketosis (anorexia, ataxia, or abnormal behavior) (9–14 d postpartum) or d 14 postpartum for control cows. Initially, a list of miRNA families and their predicted target genes for Bos taurus were downloaded from the Microcosm targets website (v. 5.0). Results for the effect of ketosis were then used to predict miRNA activity from the mRNA expression profiles through 3 approaches: Wilcoxon rank test, Ranked Ratio, and Mean absolute expression. After overlapping the results of the 3 approaches, a total of 7 miRNA were predicted as possible components in the transcriptomic response associated with ketosis and fatty liver: miR-101, miR-142, miR-186, miR-200b, miR-200c, miR-218, and miR-369–3p. To identify miRNA functions, the dynamic impact approach was then used for biological pathway analysis on the compiled differentially expressed target genes of the predicted miRNA. Pathway analysis relevant to fatty liver revealed these miRNA may play a role in fatty acid metabolism (miR-186, -200b and c, -218, -369), oxidative phosphorylation (all miRNA), peroxisome (miR-186, -200b and c), gluconeogenesis (miR-186, -369), PPAR signaling pathway (miR-101, -142, -186,-200b and c), insulin signaling pathway (miR-186, -200b, -218, -369), and apoptosis (miR-142, -200b, -369). Overall, the in silico analysis suggests that specific miRNA may be involved in the etiology of fatty liver through the control of key biological pathways and genes related to this disease. Further studies should be performed to verify the expression of these miRNA along with mRNA and protein expression of the target molecules.