Our objectives were to determine the effects of heifer age and ruminal phase (ruminal fluid: RF; ruminal solid: RS) on rumen microbial ecology and identify when heifers become faunated with ciliate protozoa. Holstein heifers (n = 12) were enrolled into an observational study at birth, managed under commercial heifer rearing practices, and followed to 21 weeks (wk) of age. A randomized complete block design with repeated measures evaluated the effects of age, phase and age × phase. Whole rumen contents were collected via esophageal intubation at 5, 9, 13, 17, and 21 wk of age. Heifers were housed in individual hutches from 0 to 9 wk (pasteurized whole milk + texturized starter), weaned at 7 wk (starter only), moved to super hutches after sampling at 9 wk (n = 3/hutch; starter+TMR), and moved to free stalls at 13 wk of age (TMR). The pen at 21 wk included 8 additional heifers without direct contact with faunated, lactating cows. Ruminal ciliate protozoa were detected in all heifers at 21 wk, but not before. Only members of the Entodiniomorphida order were observed. Ruminal ammonia decreased (P < 0.01) from 5 (2.37 ± 0.28 mM) to 9 wk (1.12 mM), and did not differ at 17 (0.91 mM) and 21 wk (1.36 mM). Bacterial community structures only differed by phase at 9 wk and were dissimilar by age and age × type (ANOSIM, P < 0.01). The number of operational taxonomic units (OTU) and Shannon diversity (OTU evenness and abundance) differed by age (P < 0.001) and the interaction of age and phase (P < 0.01). RF Shannon diversity (2.53 vs. 5.22) differed at 5 and 21 wk. RF OTU increased linearly and cubically (P < 0.01) by age: 5 wk (218), 9 wk (191), 13 wk (582), 17 wk (1029), 21 wk (1562). Relative abundance of Fibrobacteres was greater in RF than RS (P = 0.02) and greatest at 17 wk (RF: 2.51%, RS:1.47% ± 0.33%). Relative abundance of Bacteroidetes linearly increased with age (P < 0.01), but did not differ by phase or age x phase. Relative abundance of Firmicutes (RF: 19.3 ± 3.2%, RS:40.4%, P < 0.001) and Proteobacteria (RF: 34.6 ± 2.8%, RS:15.8%, P < 0.001) only differed by phase at 9 wk. Our findings suggest that heifers become faunated at 21 wk and rumen microbial ecology differs by heifer age and rumen phase.