The aim of this study was to analyze the effect of fat and protein supplementation to dairy cattle rations on milk fat triacylglycerol (TAG) composition, fatty acid (FA) positional distribution in the TAG structure, and milk solid fat content (SFC). Fifty-six lactating Holstein-Friesian cows were blocked into 14 groups of 4 cows and randomly assigned 1 of 4 dietary treatments fed for 28 d: (1) low protein, low fat, (2) high protein, low fat, (3) low protein, high fat, and (4) high protein, high fat. The high protein and high fat diets were obtained by isoenergetically supplementing the basal ration (low protein, low fat) with rumen-protected soybean meal and rumen-protected rapeseed meal, and hydrogenated palm FA (mainly C16:0 and C18:0), respectively. Fat supplementation modified milk TAG composition more extensively compared with protein supplementation. Fat supplementation resulted in decreased concentrations of the low molecular weight TAG carbon number (CN) 26 to CN34 and medium molecular weight TAG CN40, CN44, and CN46, and increased concentrations of CN38 and the high molecular weight TAG CN50 and CN52. Increased contents of C16:0, C18:0, and C18:1cis-9 in TAG in response to fat supplementation were related to increases in the relative concentrations of C16:0 and C18:0 at the sn-2 position and C18:0 and C18:1cis-9 at the sn-1(3) positions of the TAG structure. Increased concentrations of high molecular weight TAG species CN50 and CN52 in response to fat supplementation was associated with increased milk SFC at 20, 25, and 30°C. Our study shows that important alterations in milk TAG composition and structure occur when feeding hydrogenated palm FA to lactating dairy cattle, and that these alterations result in an increased SFC of milk fat. These changes in milk SFC and TAG composition and structure may improve absorption of both fat and minerals in milk-based products for infants and may affect processing of milk fat.