Aims: This study was to investigate the effect of different ratios of glucogenic to lipogenic nutrients on rumen fermentation and the corresponding ruminal bacterial communities. Methods and Results: Four diets, including glucogenic diet (G), lipogenic diet (L), two mixed diets: GL1 (G: L = 2 : 1) and GL2 (G:L = 1 : 2), served as substrates and were incubated with rumen fluid in vitro. The results revealed that the gas production, dry matter digestibility and propionate proportion were significantly increased by the G diet than others. The G diet increased the bacterial genera of Succinivibrionaceae_UCG_002, Succinivibrio, Selenomonas_1 and Ruminobacter but decreased some cellulolytic bacteria including the Eubacterium and several genera in family Ruminococcaceae than others. Conclusions: When the glucogenic nutrient was above 1/3 of the dietary energy source among the four diets, the in vitro incubation had a higher feed digestibility and lower acetate to propionate ratio. Bacterial genera, including Selenomonas, Succinivibrio, Ruminobacter, certain genera in Ruminococcaceae, Christensenellaceae_R-7_group and Eubacterium, were more sensitive to the glucogenic to lipogenic nutrients ratio. Significance and Impact of the Study: The present study provides a new perspective about the effect of dietary glucogenic to lipogenic ingredient ratios on rumen metabolism by comparing end-products, gas production and bacterial composition via an in vitro technique.