The aim of this study was to evaluate the effects of dietary Acacia mearnsii tannin extract (TE) and managing the grazing schedule, on in vitro fermentative activity of the rumen inoculum (RI) collected from dairy cows. The trial was conducted with 9 Holstein cows averaging 197 ± 12 d in milk, in a triplicate 3 × 3 Latin square design, through three 22-d experimental periods. The treatments consisted of morning grazing and afternoon TMR (AM), morning grazing and afternoon TMR added with 15 g/kg of TE (AMt), or morning TMR and afternoon grazing (PM). Cows were fed twice a day at 0700 and 1600 h, having access to TMR or pasture during 5 h. After 20 d of adaptation, RI of each cow was collected twice a day, 4 h after the morning and afternoon meal. The pH was measured and the fermentative activity of the RI was estimated through a 96-h in vitro gas-production assay. The substrates used were 2 whole-crop oat silages incubated in triplicate. Data were fitted to a simple exponential model with lag. Statistical analysis was carried out using the PROC MIXED of SAS, where treatments were compared through contrast analysis. The contrast tested were AM vs. AMt and AM vs. PM. There was not any significant interaction between treatments and time of sampling. The pH of the RI collected in the morning was lower in PM than AM (6.0 vs. 6.4; P < 0.01). For this RI, the treatments did not affect the volume of gas production (a = 243 mL/g of OM) or the rate of gas production (kd = 3.2%/h). The pH of RI collected in the afternoon was similar for all treatments (6.1). This RI collected from AMt and PM resulted in lower a compared with AM (234 and 232 vs. 246 mL/g of OM; P < 0.01), whereas no effect of treatments on the kd was observed (4.2%/h). Independently of treatments, the RI collected in the afternoon presented lower pH (P < 0.05) and higher kd (P < 0.01) than RI collected in the morning (6.1 vs. 6.3 and 4.2 vs. 3.2%/h respectively). It was concluded that tannins and afternoon grazing affects the fermentative activity of RI collected from dairy cows. The effects of sampling schedule on pH and rate of gas production suggest a higher fermentative activity of RI collected in the afternoon.