Rumen ciliates are important components of rumen microorganisms and play an important role in maintaining rumen microecology and supplying energy to the host animal. [Objectives] This study aims to clarify rumen ciliate species diversity in Holstein cows. [Methods] In this study, rumen fluid samples of five healthy mid-lactation Chinese Holstein (Bos taurus) cows were collected. For each cow, three samples were collected, and each sample includes 2 ml of rumen fluid which were fixed and stained with 8 ml MFS (methyl-green-formalin-saline) solution. The density of rumen ciliates was counted by hemocytometer: The sample was gently shaken repeatedly, and 6.5 μl of samples were immediately aspirated onto a hemocytometer plate (size: XB.K.25) to fill the counting cell, covered with a clean coverslip for 5-10 min, and the density of rumen ciliates was counted under a 10 × 10 microscope (NEXCOPE NE610, USA). Each sample was randomly counted 6 times. And the means and standard deviation of the acquired data were calculated. Total genera and percentage composition of genera of rumen ciliates were counted under light microscopy: The sample was gently shaken repeatedly, 20 μl was pipetted onto a clean slide with coverslip and 300 rumen ciliates cells were counted under a 10 × 40 microscope. The number of rumen ciliates of different genera was recorded. Each sample was randomly counted 3 times. The percentage composition of genera (%) was obtained by counting the number of each genus out of the total 300 rumen ciliates in each count and then the mean and standard deviation were calculated; the frequency of detectable genera (%) was calculated from the frequency of each genus in the total times of counts. We observed and described the common genera of rumen ciliates in the rumen of Chinese Holstein (Fig. 1), aiming to understand the rumen ciliates biodiversity [Results] The results show that the average density of ciliates in rumen fluid of Chinese Holstein was (3.2 ± 3.4) × 108 ind/L, and 13 genera of rumen ciliates were identified. Table 1 shows the results of density (× 108 ind/L) and total genera of rumen ciliates in five Chinese Holsteins. Table 2 reveals the results of the frequency of detectable genera (%) and percentage composition of genera (%) in five Chinese Holsteins. Of which three genera with the highest frequency of detection were Entodinium, Eremoplastron and Diplodinium, and three genera with the lowest frequency of detection were Elytroplastron, Metadinium and Polyplastron. In terms of the percentage composition of genera, Entodinium was the most abundant genus in the rumen fluid of Chinese Holstein, with an average percentage composition of 84.0% ± 10.2%, while the percentage compositions of all other genera were less than 5%. In Table 3 and Table 4, we also compared the results in the present study with reported data on rumen ciliates of Holstein cattle. [Conclusion] Our results reveal that rumen ciliates in rumen fluid of Chinese Holstein have rich species diversity.