In the dairy cow, late gestation and early lactation are characterized by a complexity of metabolic processes required to maintain homeostasis. The metabolome, representing the terminal downstream product of the genome, transcriptome, and proteome provides a direct measure of physiological changes in a particular condition. The objective of this study was to characterize skeletal muscle metabolome in the context of metabolic changes occurring during the transition from late pregnancy to early lactation in dairy cows. Twenty-one German Holstein cows receiving 100 g/d conjugated linoleic acid (CLA; n = 11; Lutrell pure, BASF, Germany) or a control fat supplement (n = 10; Silafat, BASF) from days in milk 1 to 182 were studied. The metabolome was characterized in M. semitendinosus samples collected at d −21, 1, 21, and 70 relative to calving using a targeted quantitative metabolomics approach (AbsoluteIDQ p180 Kit; Biocrates Life Sciences AG, Austria). Multivariate analysis of the data (PCA and PLS-DA) did not reveal separation between CLA and control animals across and within time points, whereas a clear separation between different time points, regardless of the treatment, was evident. Furthermore, ANOVA-simultaneous component analysis on the metabolites showed only time effect (P = 0.04). Out of 188 metabolites, 42 were identified as the most relevant (variable importance of projection score >1 and P < 0.05) for the observed separation. Metabolic pathway analysis identified 4 pathways that were significantly (false discovery rate <0.10) impacted over time, based on the levels of significant annotated metabolites. Out of these 4, there were 3 AA metabolism pathways including Arg and Pro metabolism, Phe, Tyr, and Trp biosynthesis, and D-Gln and D-Glu metabolism significantly affected by time along with aminoacyl-tRNA biosynthesis, which prepares for translation. In addition, long-chain fatty acylcarnitine species (C16 and C18:1) were elevated around parturition, reflecting an enhanced but incomplete mitochondrial oxidation of fatty acids. These data contribute toward a better understanding of the skeletal muscle metabolic adaption in dairy cows during the transition period.