A transcriptomics approach was used to evaluate the influence of forage-to-concentrate ratio (F:C) on liver (LV) and mammary (MG) tissue metabolism in mid-lactation dairy cows. Twenty-four primiparous lactating Holstein cows (body weight, 558 ± 10 kg; days in milk, 136 ± 37; daily milk yield, 21.12 ± 2.30 kg) were randomly allocated to 2 groups receiving either a high-forage diet (HF, F:C = 60:40) with Chinese wild rye, alfalfa hay and corn silage as forage source or a low-forage diet (LF, F:C = 40:60) with corn stover as forage source. A subgroup of cows (n = 5/diet) was used for analysis of liver and mammary transcriptome. Biopsies of LV and MG were collected at the end of 8-week feeding to measure mRNA profiles using a 4 × 44K Bovine Agilent microarray chip. Data were analyzed with SAS JMP Genomics using ANOVA with a false discovery rate correction (FDR <0.05). The Dynamic Impact Approach was used for pathway analysis. The analyses uncovered 759 and 225 differentially expressed genes (DEG) between LF and HF group in MG and LV, respectively. The greater number of DEG in MG underscored that mammary transcriptome was more responsive to changes in F:C compared with LV. Among the LV DEG, more genes were upregulated (135 vs. 90), whereas more DEG were downregulated (315 vs. 444) in MG, suggesting a potentially different response upon changes in F:C. Pathway analysis revealed enhanced amino acid metabolism, such as taurine and hypotaurine metabolism, and decreased protein export in MG of LF-fed cows. Similarly, LF-fed cows also had enhanced lipid metabolism, such as steroid hormone biosynthesis in the MG compared with cows in HL. In contrast, compared with cows in HF, LF cows had overall lower folate biosynthesis which contributed to overall lower cofactor and vitamin metabolism. Although pathway analysis revealed that amino acid, lipid, energy, as well as cofactors and vitamin metabolism were among the most impacted biological processes in MG, similar alterations were not observed in LV. Overall, results indicate that MG and LV transcriptome in primiparous dairy cows was affected to a different extent by forage-to-concentrate ratio in mid-lactation.