The objective of this study was to test the effects of essential fatty acids (EFA), particularly α-linolenic acid (ALA), and conjugated linoleic acid (CLA) supplementation on metabolic and endocrine traits related to energy metabolism, including the somatotropic axis, in mid-lactation dairy cows. Four cows (126 ± 4 d in milk) were used in a dose-escalation study design and were abomasally infused with coconut oil (CTRL; 38.3 g/d; providing saturated fatty acids), linseed and safflower oils (EFA; 39.1 and 1.6 g/d; n-6:n-3 FA ratio = 1:3), Lutalin (CLA; cis-9,trans-11 and trans-10,cis-12 CLA, 4.6 g/d of each), or EFA and CLA (EFA+CLA) for 6 wk. The initial dosage was doubled twice after 2 wk, resulting in 3 dosages (dosages 1, 2, and 3). Each cow received each fat treatment at different times. Cows were fed with a corn silage-based total mixed ration providing a low-fat content and a high n-6:n-3 fatty acid ratio. Plasma concentrations of metabolites and hormones (insulin-like growth factor-binding proteins only on wk 0 and 6) were analyzed at wk 0, 2, 4, and 6 of each treatment period. Liver biopsies were taken before starting the trial and at wk 6 of each treatment period to measure hepatic mRNA abundance of genes linked to glucose, cholesterol and lipid metabolism, and the somatotropic axis. The changes in the milk and blood fatty acid patterns and lactation performance of these cows have already been published in a companion paper. The plasma concentration of total cholesterol increased with dosage in all groups, except CLA, reaching the highest levels in EFA+CLA and CTRL compared with CLA. The high-density lipoprotein cholesterol plasma concentration increased in CTRL and was higher than that in EFA and CLA, whereas the concentration of low-density lipoprotein cholesterol increased in a dose-dependent manner in EFA and EFA+CLA, and was higher than that in CLA. Hepatic mRNA expression of 3-hydroxy-3-methyl-glutaryl-CoA synthase 1 was upregulated in all groups but was highest in EFA+CLA. Expression of sterol regulatory element-binding factor 1 tended to be lowest due to EFA treatment, whereas expression of long chain acyl-CoA-synthetase was lower in EFA than in CTRL. Hepatic mRNA expression of GHR1A tended to be higher in EFA+CLA than in CTRL. The plasma concentration of insulin-like growth factor I increased in CLA, and the plasma IGFBP-2 concentration was lower in EFA+CLA than in CTRL at wk 6. The plasma concentration of adiponectin decreased in EFA+CLA up to dosage 2. Plasma concentrations of albumin and urea were lower in CLA than in CTRL throughout the experimental period. Supplementation with EFA and CLA affected cholesterol and lipid metabolism and their regulation differently, indicating distinct stimulation after the combined EFA and CLA treatment. The decreased IGFBP-2 plasma concentration and upregulated hepatic mRNA abundance of GHR1A in EFA+CLA-supplemented cows indicated the beneficial effect of the combined EFA and CLA treatment on the somatotropic axis in mid-lactation dairy cows. Moreover, supplementation with CLA might affect protein metabolism in dairy cows.