The objective of this study was to determine the effects of dietary different rumen protected fat sources on performance and milk fatty acid composition of dairy cattle. Twenty-four multiparous Chinese Holstein were distributed into three groups with 8 cattle in each group. Group 1 were fed a basal diet consisting of corn silage, alfalfa hay and concentrate supplement; groups 2 and 3 were fed the basal diet supplemented with 200 g rumen protected unsaturated fat and rumen protected saturated fat, respectively. The pretrial period lasted for 7 d and trial period lasted for 60 d. The results showed as follows: 1) compared with group 1, supplementation of different rumen protected fat sources had no effect on dry matter intake, yield of milk, milk fat rate, yield of milk fat, yield of milk protein and feed conversion rate (P>0.05), but the milk protein rate and somatic cell counts in groups 2 and 3 were significantly higher and lower than those in group 1 (P<0.05). 2) The serum fatty acid triglyceride lipase (ATGL) activity in group 3 in the early stage was significantly higher than that in groups 1 and 2 (P<0.05). The serum ATGL activity in group 3 in the later stage was significantly higher than that in group 2 (P<0.05), and there was no significant difference among other groups (P>0.05). The activities of serum acetyl-CoA carboxylase, carnitine palmitoyl transferase-Ⅰ, aspartate aminotransferase and alanine transaminase had no significant difference among groups (P>0.05). 3) The contents of serum insulin and insulin-like growth factor-Ⅰ in the early stage in groups 2 and 3 were significantly higher than those in group 1 (P<0.05). The serum insulin content in the later stage in group 2 was significantly higher than that in groups 1 and 3 (P<0.05), and there was no significant difference in other hormone indexes among groups (P>0.05). 4) The contents of serum β-hydroxybutyrate and non-esterified fatty acid in early stage were significantly decreased by adding rumen protected fat (P<0.05), the serum malonaldehyde content in later stage was significantly decreased (P< 0. 05), the other serum biochemical indices were not affected significantly (P > 0.05). 5) Supplementation of different rumen protected fat sources alleviated body condition score decline (P< 0.05). 6) The contents of C18∶1n9c, C18∶2n6c and total unsaturated fatty acids in milk in group 2 in later stage were significantly increased compared with the other groups (P < 0. 05). While the total saturated fatty acid content was significantly decreased compared with group 3 (P<0.05), and the contents of C16∶0 and total saturated fatty acid in group 3 were significantly increased compared with other groups (P<0.05), and the other milk fatty acid contents had no significant difference among other groups (P>0.05). Overall, this study indicates that supplementation of rumen protected fat can elevate milk protein rate, reduce the content of malondialdehyde in serum, alleviate body condition score, the supplementation of unsaturated fat positively impact the content of oleic acid and linoleic acid in milk and improve the quality of milk.