Simple SummaryDiets with a lower dietary cation-anion difference could prevent hypocalcemia, enhance health, and extend the economic life of transition mammary animals. However, there is less information on rumen fermentation, cellulolytic bacteria populations, and microbiota for female goats fed a negative dietary cation-anion difference diet. We speculate that a negative dietary cation-anion difference would not affect the rumen fermentation parameters. Therefore, the present study was conducted to evaluate the effect of a negative dietary cation-anion difference diet on rumen pH, buffering capability, volatile fatty acids of acetic acid, propionic acid, butyric acid, total volatile fatty acid and acetic acid/propionic acid profiles, ruminal cellulolytic bacteria populations, and microbiota. These results provide a further evaluation on the feasibility of feeding a negative dietary cation-anion difference diet to goats.The dietary cation-anion difference (DCAD) has been receiving increased attention in recent years; however, information on rumen fermentation, cellulolytic bacteria populations, and microbiota of female goats fed a negative DCAD diet is less. This study aimed to evaluate the feasibility of feeding a negative DCAD diet for goats with emphasis on rumen fermentation parameters, cellulolytic bacteria populations, and microbiota. Eighteen female goats were randomly blocked to 3 treatments of 6 replicates with 1 goat per replicate. Animals were fed diets with varying DCAD levels at +338 (high DCAD; HD), +152 (control; CON), and −181 (low DCAD; LD). This study lasted 45 days with a 30-d adaption and 15-d trial period. The results showed that the different DCAD levels did not affect the rumen fermentation parameters including pH, buffering capability, acetic acid, propionic acid, butyric acid, sum of acetic acid, propionic acid, and butyric acid, or the ratio of acetic acid/propionic acid (p > 0.05). The 4 main ruminal cellulolytic bacteria populations containing Fibrobacter succinogenes, Ruminococcus flavefaciens, Butyrivibrio fibrisolvens, and Ruminococcus albus did not differ from DCAD treatments (p > 0.05). There was no difference in bacterial richness and diversity indicated by the indices Chao, Abundance Coverage-based Estimator (Ace), or Simpson and Shannon, respectively (p > 0.05), among 3 DCAD levels. Both principal coordinate analysis (PCoA) weighted UniFrac distance and unweighted UniFrac distance showed no difference in the composition of rumen microbiota for CON, HD, and LD (p > 0.05). At the phylum level, Bacteroidetes was the predominant phylum followed by Firmicutes, Synergistetes, Proteobacteria, Spirochaetae, and Tenericutes, and they showed no difference (p > 0.05) in relative abundances except for Firmicutes, which was higher in HD and LD compared to CON (p < 0.05). At the genus level, the relative abundances of 11 genera were not affected by DCAD treatments (p > 0.05). The level of DCAD had no effect (p > 0.05) on growth performance (p > 0.05). Urine pH in LD was lower than HD and CON (p < 0.05). Goats fed LD had higher plasma calcium over HD and CON (p < 0.05). In summary, we conclude that feeding a negative DCAD has no deleterious effects on rumen fermentation and rumen microbiota and can increase the blood calcium level, and is therefore feasible for female goats.