The purpose of the experiment was to determine if the composition of microbial inoculum affects the ruminal environment and performance of post-weaned dairy calves. Holstein bull calves (n = 20) were housed individually with sand bedding and provided texturized calf starter (19.1 ± 0.8% CP, 19.5 ± 3.9% aNDF, 39.0% ± 2.3% starch). Calves were randomly assigned to one of 4 treatments administered by stomach intubation at 3 to 6 wk of age with responses analyzed at 9 weeks of age as a randomized complete block design. Treatments included: Control: 50 mL cell-free rumen fluid (RF), bacterial-enriched RF (BE), ciliate protozoa-enriched RF (PE), or 50 mL each of BE and PE (BE+PE). A rumen contents composite from 4 cannulated Holstein cows was used to prepare the inocula either by differential centrifugation (BE; 1.4 × 10¹⁰ bacterial 16S rRNA gene copies/mL RF) or by gravimetric sedimentation (PE; 5.0 × 10⁵ protozoa/mL). Calves were killed at 9 wk of age. The BE-treated calves had greater gains in heart (28 vs 24 ± 1.0 cm, P = 0.02) and paunch girths (51 vs. 44 ± 1.7 cm, P < 0.01) between 1 and 9 weeks of age. Body weight gain (56.6 ± 2.7 kg), DMI (3.2 ± 0.1 kg/d), total ruminal DM (0.98 ± 0.12 kg), reticulorumen (7.9 ± 0.6 kg), and abomasum (1.3 ± 0.1 kg) weights did not differ by treatment. Mean ruminal protozoa densities evaluated by real-time PCR for control, BE, PE, and BE+PE treated calves were 1.1, 13.5, 8.9, and 24.1 cells/mL RF, respectively. Calves dosed with PE had lower butyrate molar proportions (5.6 vs. 8.0 ± 0.7%, P = 0.02) and tended to have greater total VFA (73.7 vs. 58.8 ± 5.0 mM, P = 0.05). Ruminal pH, ammonia and total amino acids did not differ by treatment. Relative abundance of Firmicutes tended to be greater in calves dosed with BE (30.1 vs. 22.4 ± 2.7%, P = 0.06), whereas Proteobacteria were less abundant in calves dosed with BE (13.7 vs. 27.9 ± 4.1%). Calves dosed with BE or PE had greater Shannon diversities (3.3 vs. 2.9 ± 0.1, P = 0.02) and operational taxonomic units (189 vs. 155 ± 7.7, P < 0.01). These findings show that prior microbial inoculation and inocula type altered the ruminal environment and animal performance of post-weaned dairy calves.