Previous research has indicated crude protein (CP) and neutral detergent fiber (NDF) concentrations are related to forage digestibility by horses. However, it is unclear if greater CP concentrations are simply correlated with higher quality forage or if CP amounts influence digestion and microbial fermentation in the hindgut by increasing nitrogen substrates to cellulolytic bacteria. The objective of this study was to determine the influence of varying amounts of CP on forage digestibility using an in vitro hindgut fermentation model. We hypothesized increasing CP presence would increase in vitro dry matter digestibility (IVDMD), NDF digestibility (NDFD), and acid detergent fiber digestibility (ADFD). Quadruplicate 0.5 g forage samples (alfalfa, bermudagrass, and orchardgrass) and cellulose (Sigma-Aldrich) were incubated at 37.5°C for 48 h in an ANKOM DaisyII Incubator. Casein (Sigma-Aldrich) was added to the digestion jars at 1 of 4 levels (no added casein, 66.1, 125.9, and 188.8 g) to represent control, NRC CP recommendations, industry-typical diets, and 3× NRC recommendations, after adjusting for 51% prececal CP digestibility. Freshly voided fecal samples were collected on 4 separate days from mature horses with ad libitum access to bermudagrass hay to serve as microbial inoculum for DaisyII runs (n = 4). Data were analyzed using ANOVA (SAS, v 9.3) as a split-plot design with the main plot as casein level and subplot as forage type with an error term of casein level × block. Means were separated using LSD comparisons. In vitro DMD decreased (P < 0.0001) as added casein increased. Control IVDMD across all forage types (47.5 ± 9.6%, mean ± SE) was greater than (P < 0.05) IVDMD for all added casein treatments (30.7 ± 13.0, 32.3 ± 13.6, 28.0 ± 11.2%, as casein level increased). Both NDFD and ADFD were greater (P < 0.0001) in control compared with casein-added treatments. In vitro DMD, NDFD, and ADFD differed (P < 0.0001) by forage type. Casein as a protein source may have affected results with this closed-system in vitro model. Further evaluations are needed to determine the relationship between protein in the equine hindgut and fiber digestion in vivo.