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The main purpose of this study was to determine the effect of a c straw or mixed diet on milk production, milk composition and the expression of genes associated with lactation in mid-lactation Chinese Holstein cows. In this study, 10 healthy Chinese Holstein cows were randomly assigned to two groups and fed with different diets respectively, c straw (CS) or mixed forage (MF) diet. CS group was fed roughage consisting of 53.8% c straw only and the forge to concentrate (F : C) ratio [dry matter (DM)] was about 40: 60. MF group was fed roughage consisting of 3.7% Chinese wildrye and 23.4% alfalfa hay, the forge to concentrate (F : C) ratio (DM) was 70: 30. All the cows were fed 8 weeks and body weight, dry matter intake, body condition score, fat, protein, lactose, milk yield, total solid and somatic cell count (SCC) were recorded. Quantitative real-time PCR (qRT-PCR) was used to analyze cow mammary gland samples representing two different diets. The results suggested that different diet types had significant effects on milk yield, lactose, milk fat, milk protein, dry matter intake and somatic cell count in dairy cows, and cows fed MF diet improved milk production and lactation performance clearly (P<0.05). In addition, mRNA expression of genes ACC, mTOR, STAT5, CSN2, PPARγ, FABP3 and PTEN in MF group was extremely significantly higher than that in CS group (P<0.05). mRNA expression of AKT1, FAS, SCD and SREBP1c in MF group was significantly higher than that in CS group (P<0.01). In summary, the milk yield and composition in mixed forage group were significantly improved than those in c straw group.