Saturated fatty acids (FA) stimulate the synthesis of ceramide which promotes insulin antagonism, lipolysis, and inflammation. We established that ceramide mediates insulin antagonism in bovine adipocytes, and feeding palmitic acid (C16:0) promotes ceramide accumulation in dairy cows. Our objective was to evaluate the relationship between circulating ceramide and (1) the plasma FA profile, and (2) measures of subcutaneous adipose tissue (AT) inflammation and lipolysis. In study 1, 11 cannulated mid-lactation Holstein cows were abomasally infused (280 g/d) C16:0 (PA; 85% C16:0), stearic acid (SA; 98% C18:0), or medium-chain triglycerides (MCT; C8:0/C10:0) for 7 d in a replicated Latin square design. In study 2, 16 early lactation Holstein cows received a corn silage-based diet supplemented with no added fat (soyhulls) or C16:0 (85% C16:0) at 1.5% of diet DM for 1–10 DIM. Blood and AT were collected post treatment. Lipids were measured using gas chromatography and mass spectrometry. Gene expression was assessed by qPCR. Data were analyzed under a mixed model, and correlation analyses were performed. In study 1, PA increased plasma C16:0 (P < 0.05), relative to SA or MCT. MCT increased plasma C10:0 (P < 0.01), relative to PA. Select ceramides tended to be positively correlated with plasma C16:0 (e.g., C16:0-ceramide; P ≤ 0.10). The majority of ceramides were inversely related to C8:0, C12:0, and cis-9 C18:1 (e.g., C8:0 vs C24:0-ceramide, r = −0.61; P < 0.05). In study 2, ceramides were positively correlated with hormone sensitive lipase activation (phosphorylation status; e.g., C18:1-ceramide, r = 0.83; P < 0.01) and body weight loss (e.g., C18:0-ceramide, r = 0.49; P < 0.05). Many plasma ceramides were positively correlated with AT CD44 expression (e.g., C22:0-ceramide, r = 0.67; P < 0.01), a macrophage receptor. Moreover, the ratio of C24:0- to C16:0-ceramide was positively correlated with AT macrophage trafficking measured by flow cytometry (i.e., CD14⁺ cells, r = 0.49; P = 0.05). We conclude that elevations in circulating ceramides develop with a modified FA profile, lipolysis and inflammation.