The objective of this experiment was to determine if dosing pre-weaned calves with enriched ruminal microbiota alters the rumen microbial environment and growth performance. Twenty Holstein bull calves were removed from their dam at birth, fed 3.8 L colostrum within 4 h after birth, and housed individually. Calves were fed pasteurized milk 3×/d from 0 to 7 weeks of age and offered a texturized calf starter ad libitum at 6 days of age. A randomized complete block design with repeated measures and a 2 × 2 factorial arrangement of treatments was used to evaluate responses. Treatments were administered by stomach intubation once per week from 3 to 6 weeks of age and included: 50 mL autoclaved rumen fluid (RF), 50 mL bacterial-enriched RF (BE), 50 mL protozoal-enriched RF (PE); or 50 mL of each BE and PE inoculum. A rumen content composite was collected from 4 rumen fistulated, lactating cows and used to create the inocula. BE inocula were microscopically confirmed to be free of ciliate protozoa before inoculation, while PE contained 2.9 ± 2.2 × 105 protozoa/mL. RF was collected from the calves once per week before 50 mL of the inoculum was administered. Animal performance (e.g., weight gain and dry matter intake) was not altered by inocula type. All calves were microscopically free of rumen ciliates before inoculum administration and calves that did not receive PE remained ciliate-free. Ciliate protozoa were observed in RF from 6, 8, and 6 PE treated calves (n = 10) at weeks 4, 5, and 6, respectively. Ruminal NH3 was lower in PE treated calves (3.3 vs. 6.8 ± 1.0 mM), while ruminal butyrate molar percent was greater in BE treated calves (10.8 vs. 8.3 ± 0.8). Rumen bacterial diversity measures did not differ by treatment at 3-6 weeks. Individual calf bacterial communities from treated calves became temporarily similar to the inocula at 4 weeks of age, but these communities diverged from the inocula at 5 weeks. This study provides new information about two types of rumen-derived inocula and insight into the challenges of directing the rumen microbial environment in the pre-weaned calf.