Background: The relationships between the postpartum subacute ruminal acidosis (SARA) occurrence and predicted bacterial functions during the periparturient period are still not clear in Holstein cows. Objectives: The present study was performed to investigate the alterations of rumen fermentation, bacterial community structure, and predicted bacterial functional pathways in Holstein cows. Methods: Holstein cows were divided into the SARA (n = 6) or non-SARA (n = 4) groups, depending on whether they developed SARA during the first 2 weeks after parturition.Reticulo-ruminal pH was measured continuously during the study period. Reticulo-ruminal fluid samples were collected 3 weeks prepartum, and 2 and 6 weeks postpartum, and blood samples were collected 3 weeks before, 0, 2, 4 and 6 weeks postpartum. Results: The postpartum decline in 7-day mean reticulo-ruminal pH was more severe and longer-lasting in the SARA group compared with the non-SARA group. Changes in predicted functional pathways were identified in the SARA group. A significant upregulation of pathway “PWY-6383” associated with Mycobacteriaceae species was identified at 3 weeks after parturition in the SARA group. Significantly identified pathways involved in denitrification (DENITRIFICATION-PWY and PWY-7084), detoxification of reactive oxygen and nitrogen species (PWY1G-0), and starch degradation (PWY-622) in the SARA group were downregulated. Conclusions The postpartum SARA occurrence is likely related to the predicted functions of rumen bacterial community rather than the alterations of rumen fermentation or fluid bacterial community structure. Therefore, our result suggests the underlying mechanisms, namely functional adaptation of bacterial community, causing postpartum SARA in Holstein cows during the periparturient period.