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Abstract Background Subacute ruminal acidosis (SARA) is characterized by a ruminal pH depression, and microbiota can also be affected by a higher acidity and/or dietary changes. Previous studies have revealed similar patterns in pH reduction in the rumen and reticulum, whereas changes in reticular pH and bacterial community following a high-grain diet are not fully understood. The aim of this study was to investigate the changes in reticular pH and bacterial community structure following a high-grain diet simultaneously with those in the rumen. Results SARA was diagnosed when ruminal and reticular pH remained under 5.6 for 350 ± 14 and 312 ± 24 min/d, respectively, on the last day of the CON period. During the CON period, lower proportion of acetic acid and higher proportion of butyric acid were observed compared with the HAY period. The proportions of acetic acid and propionic acid were lower and higher, respectively, in the rumen compared with the reticulum. From 454 pyrosequencing analysis, the relative abundance of several genera differed significantly between the two periods and the two locations. During the HAY period, higher relative abundances of Prevotella, Eubacterium, Oscillibacter, and Succiniclasticum and lower relative abundances of Ruminococcus, Clostridium, and Olsenella were identified compared with the CON period. Furthermore, the relative abundance of Eubacterium was lower in the rumen compared with the reticulum. Bacterial diversity indices were significantly different between the HAY and CON periods, being higher in the HAY period. The quantitative real-time PCR showed that the copy numbers of several cellulolytic bacteria (Fibrobacter succinogenes and Ruminococcus albus) were higher during the HAY period. Conclusion A high-grain diet showed similar impacts on the pH, fermentation, and bacterial community structure in the rumen and reticulum. During the CON period, ruminal and reticular pH decreased following the high-grain challenge, and lower bacterial diversity and changes in the bacterial composition, similarity, and bacterial copy numbers were observed due to a higher acidity and dietary changes compared with the HAY period. These changes may influence the fermentative ability of the rumen and reticulum.