Phospho-proteomics is a new frontier in omics-research that reveals the full repertoire of phosphorylation sites in proteins. Conjugated linoleic acid (CLA) supplementation reduces energy output in milk, which could increase fat storage in transition cows, but it is also known to promote lipolysis in adipose tissue (AT). Our objective was to elucidate the effect of CLA on protein activation in AT of transition cows by phospho-proteomics. Ten rumen cannulated Holstein cows were fed a corn silage-based TMR with low fat content and daily abomasally supplemented from wk 9 prepartum until slaughtering at 63 d postpartum with coconut oil (CTL, n = 5; 45.5% C12:0; 16.9% C14:0; 76g/d) or Lutalin (CLA, n = 5; c9, t11 and t10, c12, 10g/d). Subcutaneous (S) and abdominal (A) AT were collected and frozen (−80°C). Proteins were extracted for global quantification of the phospho-proteome by enrichment of phospho-peptides (PP) by Fe immobilized metal ion affinity chromatography (Fe-IMAC) followed by discovery analysis. Data were analyzed for the effect of fat depot (S vs. A), treatment (CLA vs. CTL), and their interaction by 2-way ANOVA, and bioinformatics analysis was conducted by Ingenuity (Qiagen). Overall, CLA cows had lower fat and energy output in milk compared with CTL based on weekly milk components, and had more total and abdominal AT than CTL (P < 0.005). A total of 5,919 PP were identified in AT. The abundance of 854 PP (14.4%) was statistically different between CLA and CTL (P < 0.05, FC ± 1.5). Increased protein phosphorylation, i.e., higher abundance of several PP, was found in lipid-metabolism proteins in CLA vs. CTL: 7 PP were more abundant in acetyl-CoA carboxylase 1 (ACACA); 9 PP in fatty acid synthase (FASN); 4 PP in hormone sensitive lipase (HSL); and 3 PP in perilipin (PLIN). Increased total abundance of FASN and HSL was found by immunoblots in CLA vs. CTL AT (P < 0.02). Top canonical pathways enriched in CLA AT were protein kinase A signaling and insulin receptor signaling. These novel findings provide insight to the molecular mechanism by which CLA might stimulate both lipogenesis and lipolysis in AT.