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Efficient nitrogen utilization in dairy cows is essential for maximizing lactation performance while minimizing nitrogen losses. This experiment was conducted to evaluate the effects of the additive W70 and varying dietary protein levels on lactation performance, rumen fermentation, blood parameters, and nitrogen metabolism in lactating dairy cows. 16 Holstein cows were utilised in a replicated 4 × 4 Latin square with 2 levels of crude protein (CP) (16 vs. 17%) and 2 levels of additive W70 (0 vs. 5 × 1010 cfu/g, 20 g/d). The cows were randomly assigned to 4 treatment groups: 16% CP no additive (16%C), 16% CP with additive W70 (16%A), 17% CP no additive (17%C), 17% CP with additive W70 (17%A). An interaction was observed between CP and additive W70. Specifically, additive W70 decreased dry matter intake (DMI) (P < 0.01), nitrogen (N) intake (P < 0.01), retained N content (P < 0.05), productive N content (P < 0.05), retained N proportion (P < 0.05), productive N proportion (P < 0.10), increased milk yield/DMI (P < 0.01) and nitrogen utilization efficiency (NUE) (P < 0.10) in the 16% CP diet, whereas no differences were observed in the 17% CP diet. The additive W70 increased fat corrected milk (FCM)/DMI (P < 0.10) and energy corrected milk (ECM)/DMI (P < 0.05), and reduced ruminal ammonia nitrogen (NH3-N) (P < 0.01), with no interaction with dietary CP level. The relative abundance of the genera Sphingomonas, Solibacillus, Desemzia, Peribacillus, Succinivibrionaceae UGG-001, Prevotella, and Succiniclasticum were higher in the rumen following the addition of additive W70 in both the 16% and 17% CP diets. Milk yield (P < 0.01), milk urea nitrogen (MUN) (P < 0.01), milk protein yield (P < 0.01), FCM yield (P < 0.05), ECM yield (P < 0.01), FCM/DMI (P < 0.10), and ECM/DMI (P < 0.05) were all increased in response to the 17% CP diet, whereas DMI and the content of milk fat, milk protein, and milk lactose were unaffected. The 17% CP diet increased the acetate to propionate ratio (P < 0.10) and NH3-N (P < 0.10), while reducing isovalerate (P < 0.10) and valerate (P < 0.10). The 17% CP diet increased blood urea nitrogen (BUN) (P < 0.01) and glucose (GLU) (P < 0.05). Cows fed a 17% CP diet had a higher milk N content (P < 0.05), fecal N content (P < 0.01), N loss content (P < 0.01), fecal N proportion (P < 0.01), urine N proportion (P < 0.10), and N loss proportion (P < 0.10) than those receiving 16% CP. The relative abundance of the genera Kandleria, Ligilactobacillus, and Escherichia-Shigella were lower in the 17% CP diet than in the 16% CP diet despite whether additive W70 was added or not. Supplementation with additive W70 improved feed conversion and NUE in a 16% CP diet, while the additive W70 modulated the rumen microbiota and reduced ruminal NH3-N. The 17% CP diet enhanced milk yield but increased N losses and altered rumen microbial composition.