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The effects of live yeast (Saccharomyces cerevisiae) and ruminal environment on in vitro true digestibility of dry matter, organic matter, and fiber components were evaluated. Ruminally-cannulated beef steers (n = 4; BW = 520 ± 30 kg) were used in a cross-over design to serve as donors of ruminal content. In vitro substrates (6 grower and 6 finisher diets) and individual ingredients (steam-flaked corn, wet corn gluten feed, and old-world bluestem hay) were dehydrated (55o C), ground (1 mm), and incubated using a 2 × 2 factorial arrangement of treatments: a) the presence or not of yeast (8×109 CFU/animal-daily adjusted to a 70 L ruminal content volume), and b) two ruminal inoculums collected from donors fed a grower (50:50) or a finisher (92:08) diet (concentrate:forage). Four incubation batches (experimental unit) were performed, in which the initial two batches were performed followed by another two after animals switched diets and were adapted again during 21d. A Daisy in vitro incubation system was used, and samples incubated for 48h (39°C). The GLIMMIX procedure of SAS was used for the statistical analysis. No 3-way interactions (P ≥ 0.71) or 2-way interactions with substrate were observed (P ≥ 0.25). The in vitro true digestibility of DM, OM, NDF, and hemicellulose increased when yeast was added to the incubator containing a 50:50 ruminal content (Rumen × Yeast; P ≤ 0.02). The ADF digestibility increased (P = 0.02) with yeast addition regardless of ruminal content type (50:50 or 92:08). As expected, finisher substrate diets were more (P < 0.01) digestible than grower diets. The combination of live yeast and a specific ruminal environment seemed to be more important than substrate type, in which live yeast improved in vitro digestion within a 50:50 ruminal inoculum more evidently.