Simple SummaryRumen microbes play an important role in the health and production of ruminants, and they are influenced by dietary changes. In our study, we investigated the change of rumen bacteria under the four treatments of dietary non-fibrous carbohydrate (NFC) to neutral detergent fiber (NDF) ratios in sheep using three generations of full-length amplifiers sequencing. As rumen is a complex organ, and the effects of dietary NFC/NDF ratio change on ruminal bacteria might change over time, thus the study was conducted for four periods of 72 d in total. The results showed that the composition of rumen bacteria changed with different dietary NFC/NDF ratio during the experimental periods. Rumen bacterial diversity was decreased in dietary NFC/NDF ratio of 1.90 with the prolong of experimental periods. The main dominant phyla in Karakul sheep rumen didn’t change, while their relative abundance changed with dietary NFC/NDF ratio and experimental periods. The relative abundance of unidentified-Lachnospiraceae and main cellulose-degrading bacteria was higher in dietary NFC/NDF ratio of 1.37 than other groups (NFC/NDF ratio of 0.54, 0.96 and 1.90).The study was conducted to investigate the effects of dietary NFC/NDF ratio change on rumen bacteria in sheep. Twelve Karakul sheep were assigned randomly into four groups fed with four dietary NFC/NDF ratios of 0.54, 0.96, 1.37, and 1.90 and they were assigned into groups 1, 2, 3, and 4, respectively. The experiment was divided into four periods: I (1–18 d), II (19–36 d), III (37–54 d), and IV (55–72 d). In each period, the first 15 d were used for adaption, and then rumen fluid was collected for 3 d from each sheep before morning feeding. The fluid was analyzed with three generations of full-length amplifiers sequencing. Results showed that the bacterial diversity of group 4 was decreased in period III and IV. At the phylum level, Bacteroidetes (37–60%) and Firmicutes (26–51%) were the most dominant bacteria over the four periods. The relative abundance of Bacteroidetes, Firmicutes, Tenericutes, and Spirochaete changed with dietary NFC/NDF ratio change over the four periods, but there was no difference among groups over the four periods (p > 0.05). At the genus level, unidentified-Lachnospiraceae was the dominant genus, and its relative abundance in group 3 was high during the period I and III (p < 0.05). The relative abundance of Mycoplasma in group 4 was high in the period I and II (p < 0.05). The relative abundance of Succiniclasticum was high in group 2 of period II (p < 0.05). At the species level, the relative abundance of Butyrivibrio-fibrisolvens was found to be high in group 3 during periods I and III (p < 0.05). The main semi-cellulose-degrading bacteria and starch-degrading bacteria were low, and there was no significant difference among groups over four periods (p > 0.05). Taken together, the dietary NFC/NDF ratio of 1.90 decreased the diversity of bacteria as a period changed from I to IV. While the main phylum bacteria didn’t change, their relative abundance changed with the dietary NFC/NDF ratio change over the four periods. The most prevalent genus was unidentified-Lachnospiraceae, and its relative abundance was higher in dietary NFC/NDF ratio of 1.37 than other groups. Similarly, the main cellulose-degrading species was higher in the treatment of dietary NFC/NDF ratio of 1.37 than other groups.