MicroRNA (miRNA) are abundant in milk, and various miRNA species regulate functions including lactation and immunity. The study objective was to determine the miRNA profile in colostrum of over-conditioned cows compared with cows of more moderate BCS at calving. Multiparous cows with either high (≥4.0; n = 7) or moderate BCS (2.75–3.50; n = 9) in the week before parturition were selected from a commercial dairy herd. Blood was sampled within 24 h after calving and analyzed for serum free fatty acids (FFA). Colostrum was collected within 20 h of calving; miRNA was isolated from colostrum samples after removing milk fat and cells. MicroRNA were sequenced and reads were mapped to the bovine genome and to the existing database of miRNA at miRBase.org. Two programs, Oasis 2.0 and miRDeep2, were employed in parallel for mapping reads to the bovine genome. Analysis of miRNA count data was performed using DEseq2. After removing miRNA with less than 10 counts across all samples, 343 and 415 known miRNA mapping to miRBase were detected from Oasis and miRDeep2, respectively. Identification of differentially expressed miRNA from DEseq2 was not affected by the differences in number of miRNA detected by the 2 mapping programs. Most abundant miRNA included miR-30a, miR-148a, miR-181a, let-7f, miR-26a, miR-21, miR-22, and miR-92a. Large-scale shifts in miRNA profile were not observed; however, colostrum of cows with high BCS contained fewer miR-486 (fold change −2.15; false discovery rate P = 0.06), which has been linked with altered glucose metabolism. Colostrum from cows with elevated FFA (greater than 700 μM) contained fewer miR-10b (fold change −2.48; false discovery rate P < 0.01), which may have regulatory effects on inflammatory signaling and lipid metabolism. These miRNA may provide a link between periparturient cow adiposity and mammary nutrient metabolism. Potential functions of abundant miRNA suggest involvement in development and maintenance of cellular function in the mammary gland, with the additional possibility of influencing neonatal tissue and immune system development.