Selection for more feed efficient dairy cows is key to improving sustainability and profitability of dairy production; however, underlying mechanisms contributing to individual animal feed efficiency are not fully understood. The objective of this study was to identify circulating metabolites, and pathways associated with those metabolites, that differ between efficient and inefficient Holstein dairy cows using targeted metabolite quantification and untargeted metabolomics. The top and bottom fifteen percent of cows (n = 28/group) with the lowest and highest residual feed intake in mid-lactation feed efficiency trials were grouped retrospectively as high-efficient (HE) and low-efficient (LE). Blood samples were collected for quantification of energy metabolites, markers of hepatic function, and acylcarnitines, in addition to a broader investigation using untargeted metabolomics. Short-chain acylcarnitines, C3-acylcarnitine, and C4-acylcarntine were lower in HE cows (n = 18/group). Untargeted metabolomics and multivariate analysis identified thirty-nine differential metabolites between HE and LE (n = 8/group), of which twenty-five were lower and fourteen were higher in HE. Pathway enrichment analysis indicated differences in tryptophan metabolism. Combined results from targeted metabolite quantification and untargeted metabolomics indicate differences in fatty acid and amino acid metabolism between HE and LE cows. These differences may indicate post-absorptive nutrient use efficiency as a contributor to individual animal variation in feed efficiency.