Twelve 5-year-old beef steers, with an average weight of 2000 lbs., fitted with rumen canulae were used in a 4 × 4 incomplete Latin square design to examine the impact of the direct fed microbial Propionibacterium acidipropionici CP 88 (PA) on rumen fermentation characteristics, in vitro CH4, CO2, and N2 production, and in vivo CH4 and CO2 production. All steers were housed in the same pen equipped with eight GrowSafe feeding stations to monitor individual animal feed intake and one GreenFeed System to estimate individual animal CH4 and CO2 production. Steers were fed a corn-silage-based diet throughout the experiment. Treatments consisted of PA administered at: (1) control (0.0); (2) 1.0 × 108; (3) 1.0 × 109; and (4) 1.0 × 1010 cfu∙animal−1∙day (d)−1. Treatments were administered directly into the rumen as a single bolus dose daily. On day 7 and 14 of each period, rumen fluid was collected from each steer 2 h post treatment administration for VFA analysis and in vitro DM digestibility determination. Following a 14 d washout period, animal treatments were switched and the experiment repeated until the 4 × 4 Latin square was complete. In vivo propionic acid molar proportions and total VFA concentrations were greater (p < 0.05) in steers receiving PA when compared with controls. All other in vivo rumen fermentation characteristics were similar across treatments. In vitro DM disappearance (p < 0.05) and total VFA (p < 0.05) were greater and CH4 lesser (p < 0.04) in fermentation vessels incubated with rumen fluid from animals receiving PA when compared with controls. Dry matter disappearance (p < 0.03) and propionic acid molar proportions increased (p < 0.04) linearly as the dose of PA increased. In vitro total VFA tended (p < 0.08) to increase linearly and CH4 production per unit of DM digested tended (p < 0.09) to decrease quadratically in response to PA dose. All other in vitro rumen fermentation characteristics were similar across treatments. These data indicate that PA impacts in vivo and in vitro rumen fermentation characteristics.