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Simple Summary: The yak is a unique species found on the Tibetan plateau, adapted to the high-altitude, low-oxygen environment, providing meat, milk, and other resources to local herders, and is an indispensable means of production and livelihood for herders on the Tibetan plateau and a major source of economic income for Tibetan herders. A change in diet leads to indigestion and anorexia during the acclimatization phase, which can lead to changes in dry matter intake and daily weight gain in the early part of the housing period. The rumen microbiota will be reshaped. Ruminal fluid transplantation can reshape the rumen microbiota of the recipient animals, which can rapidly restore the microbiota to a new homeostasis. Considering the special characteristics of yaks on the Tibetan plateau, the dynamic seasonal changes in pasture and the trend of changing from natural grazing to confinement farming, this study focused on the changes in rumen flora due to dietary changes and the improvement of rumen fluid transplantation on the remodeling of yak flora and production performance when yak breeding patterns were changed. A relatively stable microbial ecological balance system in the rumen plays an important role in rumen environment stability and ruminant health maintenance. No studies have reported how rumen fluid transplantation (RFT) affects the composition of rumen microorganisms and yak growth performance. In this experiment, we transplanted fresh rumen fluid adapted to house-feeding yaks to yaks transitioned from natural pastures to house-feeding periods to investigate the effects of rumen fluid transplantation on rumen microbial community regulation and production performance. Twenty yaks were randomly divided into the control group (CON; n = 10) and the rumen fluid transplantation group (RT; n = 10). Ten yaks that had been adapted to stall fattening feed in one month were selected as the rumen fluid donor group to provide fresh rumen fluid. Ruminal fluid transplantation trials were conducted on the 1st, 3rd, and 5th. Overall, 1 L of ruminal fluid was transplanted to each yak in the RT and CON group. The formal trial then began with both groups fed the same diet. After this, growth performance was measured, rumen fluid was collected, and rumen microbial composition was compared using 16s rRNA sequencing data. The results showed that rumen fluid transplantation had no significant effect on yak total weight gain or daily weight gain (p > 0.05), and feed efficiency was higher in the RT group than in the CON group at 3 months (treatment × month: p < 0.01). Ruminal fluid transplantation significantly affected rumen alpha diversity (p < 0.05). Up to day 60, the RT group had significantly higher OTU numbers, Shannon diversity, and Simpson homogeneity than the CON group. Principal coordinate analysis showed that the rumen microbiota differed significantly on days 4 and 7 (p < 0.05). Bacteroidota, Firmicutes, Proteobacteria, and Spirochaetes were the most abundant phyla in the rumen. The relative abundances of Bacteroidota, Proteobacteria, and Spirochaetes were lower in the RT group than in the CON group, with a decrease observed in Bacteroidota in the RT group on days 7 and 28 after rumen fluid transplantation (p = 0.013), while Proteobacteria showed a decreasing trend in the CON group and an increasing trend in RT; however, this was only at day 4 (p = 0.019). The relative abundance of Firmicutes was significantly higher in the RT group than in the CON group on days 4, 7, and 28 (p = 0.001). Prevotella and Rikenellaceae_RC9_gut_group were the predominant genera. In conclusion, our findings suggest that rumen fluid transplantation improves yak growth performance and rumen microbial reshaping. The findings of this study provide new insights into yak microbial community transplantation and a reference for improving feed efficiency in the yak industry. [ABSTRACT FROM AUTHOR]