The effects of a saponin-based surfactant on tempered barley grain were studied in three in vitro experiments. In Experiment 1, barley kernels were tempered at 220 or 246 g/kg moisture with surfactant included at 0, 60 or 120 μl/kg dry matter (DM). Surfactant increased ( P < 0.05) the rate of water uptake during the initial 2 h of tempering, but not if tempering was extended beyond 4 h. Experiment 2 evaluated effects of surfactant on barley grain processing characteristics. Surfactant was included at 0, 60, 120, or 240 μl/kg DM during tempering of barley (190 g/kg moisture; 2 or 4 h), then the barley was processed with rollers set 2.032, 2.223 or 2.413 mm apart. Surfactant did not affect ( P > 0.05) the processing index (PI) of the barley or the particle size distribution. Increasing the roller distance from 2.032 to 2.413 mm increased ( P < 0.05) the PI and the proportion of particles with 3.35–4.75 mm diameter, and reduced ( P < 0.05) the proportions of particles larger than 4.75 mm or smaller than 2.36 mm. No surfactant × roller distance interactive effects on processing characteristics were observed. In Experiment 3, barley was tempered (190 g/kg moisture; 2 h) with surfactant at 0 or 120 μl/kg DM and processed with rollers at 2.032 or 2.413 mm, then incubated in batch culture with surfactant supplemented at 0 or 360 μl/kg DM. Apparent in vitro dry matter digestibility (IVDMD) and gas production were higher ( P < 0.01) with rollers at 2.032 mm than at 2.413 mm. Irrespective of substrate particle size, applying surfactant during tempering and/or prior to incubation reduced ( P < 0.05) the 4 h IVDMD, accumulation of volatile fatty acids (VFA) and reducing sugars, and molar percentage of acetic acid in the VFA, but it increased ( P < 0.05) the molar percentage of butyric acid. The acetate:propionate ratio at 4 h was reduced ( P < 0.05) by surfactant applied during (120 μl/kg DM) and/or after (360 μl/kg DM) tempering if the barley had been rolled at 2.032 mm, but at 2.413 mm roller spacing this effect on acetate:propionate was observed only with the maximum application of surfactant (120 + 360 μl/kg DM). Despite enhancing the initial rate of water absorption by barley kernels, surfactant applied during tempering did not affect the processing characteristics of barley. Added during or after tempering, surfactant reduced the extent of IVDMD measured at 4 h, but not at 24 h.