AimsTo evaluate the effects of condensed tannins (CTs) fractions of differing molecular weights (MWs) from a Leucaena leucocephala hybrid-Rendang on the rumen protozoal community invitro. Methods and ResultsThe effects of unfractionated CTs (F0) and CT fractions of different MWs (F1>F2>F3>F4>F5) on protozoal population and community were evaluated invitro using rumen microbes and ground guinea grass as the substrate. Higher-MW CT fractions F1 and F2 significantly (P<005) decrease the number of ciliate protozoa. The real-time PCR analysis showed that the total protozoa was significantly (P<005) lower in F0 and all CTs with fractions F1 and F2 having the lowest value. High-throughput sequencing of the partial 18S rRNA gene showed that the genus Entodinium significantly (P<005) decreased with increasing MWs of CT, whereas Anoplodinium-Diplodinium were significantly (P<005) increased. Inclusion of the highest MW CT fraction F1 decreased the relative abundance of the minor genera such as Eudiplodinium and Polyplastron compared to the control and CT fractions F2-F5. ConclusionCTs of differing MWs could reduce and alter the rumen protozoa population in vitro. This effect was more pronounced for higher-MW CTs. Significance and Impact of the StudyThe high MW CTs should be considered as a feed supplement in the ruminant diet to reduce the protozoal population which are known to be associated with methanogens as a means to mitigate methane production in the rumen.