Few studies have examined the effects of puerarin (PE) on ruminant parameters and methane production. Therefore, we determined the degradation of PE in the rumen and evaluated the effect of PE on in vitro fermentation, methanogenesis, and microbial community structure. A completely randomized design was used for the in vitro fermentation, and 4 gradient dosages of PE (0 mg/kg, 50 mg/kg, 100 mg/kg, and 150 mg/kg of DM) were applied in this trial. The in vitro fermentation was carried out in three runs at 6 h and 48 h, with four replicates per treatment per time point. Each run included 40 samples: eight treatments × four replicates and eight blank samples. Based on the fermentation results, both the PE treatments and the control group (CON) at 48 h were chosen for further analysis to explore the effects of PE on the bacterial community structure. Meanwhile, we determined the degradation rate and degradation products of PE in vitro ruminal fluid using high-performance liquid chromatography (HPLC). In this trial, PE may be isomerized into daidzin by rumen microorganisms; the in vitro degradation results of PE indicated that 70% of PE was degraded within 6 h, with the degradation rate reaching nearly 85% by 12 h. The concentrations of NH₃-N and microbial crude protein (MCP) significantly increased linearly with the PE doses at 6 h (p = 0.01). The concentrations of MCP (p = 0.02) and propionate (p = 0.04) demonstrated a linear increase with increasing PE doses at 48 h. In contrast to microbial protein (MCP) and propionate, the acetate-to-propionate ratio decreased linearly with increasing PE doses at 48 h (p = 0.05). Additionally, the addition of PE linearly decreased methane production at 48 h (p = 0.01). Meanwhile, the relative abundances of g_UBA1217 (p = 0.03), g_UBA2810 (p = 0.04), and g_Succiniclasticum (p = 0.03) were significantly lower compared with the CON group. The results showed that PE can be degraded by rumen microflora. Furthermore, it can improve rumen fermentation parameters, increase the amount of protein synthesized by rumen microorganisms, and reduce methane production and the acetate-to-propionate ratio. PE could potentially be an effective strategy for methane mitigation; however, further research is needed to assess its in vivo effects in dairy cows over a longer period.