Technical Abstract: Leafy spurge (LS; Euphorbia esula) is indigenous to Eurasia and is rapidly changing the landscape in the northern Great Plains and Intermountain West. Sheep consume LS at a higher rate than cattle. Our objectives were to investigate LS in vitro digestibility and gas production by bovine vs ovine inoculum with or without LS in the diet. Two phases were used with two ruminally cannulated cows and ewes as rumen fluid donors. Each phase allowed for 7 d adaptation to diets. Phase 1 consisted of animals fed a diet of (DM basis) 15% LS (21.9% CP, 48% NDF, DM basis) and 85% barley hay (BH; 12% CP, 56%NDF, DM basis) based on previous day intake. Phase 2 animals were fed 100% BH. Substrates for in vitro digestion and gas production consisted of alfalfa or LS:BH ratio in 10% increasing increments for a total of 12 treatments. Gas production extent (mL) and rate (mL/h) were calculated after 96 h of incubation. There was a LS exposure×treatment interaction (P<0.01) observed for extent of gas production. Extent of gas production with increasing LS was reduced by 9% when animals were exposed to LS and by 11% with no LS exposure. A species × LS exposure interaction for rate of gas production (P=0.02) was observed. Exposure to LS decreased rate of gas production by 5% for bovine compared to ovine. However, no exposure to LS caused the opposite effect with a 5% increase in bovine rate of gas production. In vitro digestion was evaluated after 48 h incubation. Model main effects of treatment and species were significant for IVDMD (P<0.01 and P=0.06, respectively). Increasing LS:BH ratio in the substrate resulted in an 18% increase in IVDMD and bovine IVDMD was higher than ovine (72.1 vs 71.3 + 0.31). Alfalfa, BH, and LS IVDMD was 66, 64, and 81%, respectively. These data indicate LS is highly digestible and prior exposure to LS may change rumen microbial ecology and ultimately influence utilization of LS in vivo.