This study investigated the effects of inulin on rumen fermentation parameters, ruminal microbiome and metabolites, as well as lactation performance and serum indexes in dairy cows. Sixteen Holstein dairy cows with similar body conditions were randomly divided into 2 groups (
n
= 8 per group), with inulin addition at 0 and 200 g/d per cow. The experiment lasted for 6 weeks, including a 1-week adaptation period and a 5-week treatment period. At the end of the experimental period, the milk, serum and rumen fluid were sampled and analyzed. The microbiome and metabolome in the rumen fluid were analyzed via 16S rRNA sequencing and untargeted metabolomics, respectively. The results showed that supplementation with inulin (200 g/d per cow) increased the milk yield (
P
= 0.001), milk protein (
P
= 0.032), lactose rate (
P
= 0.004) and proportion of saturated fatty acids (SFA) in milk (
P
< 0.001), but decreased the proportion of unsaturated fatty acids (USFA) (
P
= 0.041). Rumen pH (
P
= 0.040) and the concentration of NH
3
–N (
P
= 0.024) were decreased; however, acetate (
P
< 0.001), propionate (
P
= 0.003), butyrate (
P
< 0.001) and lactic acid (LA) (
P
= 0.043) were increased. The total cholesterol (TC) (
P
= 0.008) and triglycerides (TG) (
P
= 0.01) in serum were also reduced. Additionally, inulin addition elevated the relative abundance of several beneficial symbiotic and short-chain fatty acid (SCFA)-producing bacteria, such as
Muribaculaceae
(false discovery rate [FDR]-adjusted
P
< 0.01),
Acetitomaculum
(FDR-adjusted
P
= 0.043), and
Butyrivibrio
(FDR-adjusted
P
= 0.036), while elevating the levels of L-lysine (FDR-adjusted
P
= 4.24 × 10
−3
), L-proline (FDR-adjusted
P
= 0.0158), and L-phenylalanine (FDR-adjusted
P
= 0.027). In contrast, several pathogens and ruminal bacteria abundant in high-fat diets, such as
Escherichia-Shigella
(FDR-adjusted
P
= 0.022),
Erysipelotrichaceae
__UCG-004
(FDR-adjusted
P
< 0.01) and
RF39
(FDR-adjusted
P
= 0.042) were decreased along with the reduction of lysophosphatidylcholine (LysoPC) (18:1 (9Z)) (FDR-adjusted
P
= 1.03 × 10
−3
), LysoPC (16:0) (FDR-adjusted
P
= 0.0108), LysoPC (18:2 (9Z, 12Z)) (FDR-adjusted
P
= 1.65 × 10
−3
) and 8-methylnonenoate. I...