This experiment was aimed to study the changes of biochemical indexes and gene expressions related to lipid metabolism in mammary tissue for hyperlipidemia dairy cows. Twelve Holstein dairy cows were selected and the main biochemical indexes in serum of dairy cows were determined by automatic biochemical analyzer. The dairy cows were divided into normal group and high lipid group according to whether the contents of total cholesterol (TC) and triglycerides (TG) in serum increased. Mammary tissue was selected for pathological sections and stained with oil red O, to observe pathological change and lipid droplet in mammary tissue. Total RNA of mammary tissue was isolated to detect the relative expression levels of lipid metabolism related genes by real time quantitive PCR (qRT-PCR). The results showed as follows:1) the lipid metabolism indexes of TC, TG and very low density lipoprotein (VLDL) contents in mammary tissue of high lipid group were significantly increased (P<0.05), the high density lipoprotein cholesterin (HDL-C) content was decreased, and the low density lipoprotein cholesterin (LDL-C) was increased, but no significant differences of them were found (P>0.05). 2)Pathological observation of dairy cows in the high lipid group showed that mammary epithelial cells shedding and mammary acinar wall was obviously thickened as well as cell swelling, some cell fat droplets increased, nuclear chromatin shrank, edge shift, cell contours unclear. Oil red O staining showed that the nuclei of dairy cows in the normal group was blue, and the red area was less, mainly concentrated on the edge of the cell, only a small amount of lipid droplets gathered. In the high lipid group, there were large red areas, and the connective tissue density increased. The lipid droplets were fused together to form large vacuolated fat cells. 3)Compared with the normal group, the relative expression levels of endogenous synthesis key enzyme gene acetyl-CoA carboxylase 1 (ACC1) and activation genes acetyl-CoA synthase 2 (ACSS2) and long chain lipid coenzyme A synthase 1 (ACSL1) of mammary fat metabolism in the high lipid group were significantly increased (P<0.05), while the relative expression levels of uptake and transport key genes fatty acid binding protein 3 (FABP3), differentiated antigen cluster 36 (CD36) and lipoprotein lipase (LPL), denaturize key genes stearyl coenzyme A desaturase (SCD) and fatty acid desaturase 1 (FADS1), lactamase key gene acetyl glycerol phosphatidyl transferase 6 (AGPAT6) were significantly reduced (P<0.05). It can be concluded from the present study that hyperlipidemia causes blood lipid level increasing and make obvious pathological changes of mammary tissue as well as mammary lipid metabolism related genes expression of dairy cows.