The objective of this study was to determine if microbial inoculum composition affects dairy calf rumen microbial ecology. Holstein bull calves (n = 20) were removed from their dam at birth and individually housed in calf hutches with sand bedding. Responses were studied using a randomized complete block design with repeated measures and a 2 × 2 factorial arrangement of treatments: stomach intubation of 50 mL of rumen fluid (RF) as autoclaved RF, bacterial-enriched RF (BE), protozoal-enriched RF (PE), or 50 mL of each BE and PE. Inocula were prepared fresh by differential centrifugation of blended RF+solids (BE) or gravimetric sedimentation of RF (PE) from a rumen content composite taken from 4 rumen fistulated, primiparous Holstein cows (mean ± SD: 101 ± 5 DIM, 37.8 ± 2.0 kg milk/d). BE was microscopically confirmed to be free of ciliates, while PE contained 2.9 ± 2.2 × 10⁵ cells/mL. Calf RF was sampled and inocula was administered on 1 d/wk during wk 3–6 of age by stomach intubation; RF sampling immediately preceded inoculation. Data were analyzed as repeated measures with BE, PE, and the interaction as contrasts of interest with P < 0.05 as significant. Calf DMI and ADG were 0.4 ± 0.08 and 0.9 ± 0.1 kg/d, respectively and not different by treatment. At 3 wk, all calves were fauna-free and calves that did not receive PE remained fauna-free through wk 6. Ciliate protozoa were observed in RF from 6, 8, and 6 PE calves (n = 10) at wk 4, 5, and 6, respectively; ciliate concentrations averaged 1.5 × 10⁴/mL in these calves. Butyrate molar percent was greater in BE calves (10.8 vs 8.3 ± 0.8), while other VFA molar percentages were not affected by treatment; ruminal NH₃ was lower in PE calves (3.3 vs 6.8 ± 1.0 mM). Rumen microbial α and β diversities (by targeted PCR of the V4 region of the 16S rRNA gene and sequencing with Illumina MiSeq) did not differ by treatment or wk. PE calves had greater relative abundances of the genus Prevotella (33 vs. 18 ± 5.3%) and phylum Proteobacteria (12.4 vs. 2.3 ± 3.2) at wk 4 and 5, respectively. These findings demonstrate the potential for inoculation with BE and PE to affect the ruminal environment.