Aflatoxin is a potent carcinogen commonly found in animal feeds that can impair rumen fermentation at high concentrations; however, its effects at physiologically relevant concentrations are unknown. This study examined the effects of aflatoxin B (AFB), with or without bentonite clay (CL) and Saccharomyces cerevisiae fermentation product (SCFP)-based sequestering agents on in vitro rumen fermentation and digestibility of a dairy cow TMR. Corn silage-based TMR (0.5 g, 17.3% crude protein and 1.67 Mcal/kg of net energy for lactation) was incubated in a rumen fluid-buffer inoculum (1:2 ratio; 50 mL) with the following treatments: (1) no additives (control); (2) control + 0.75 µg/L of AFB1 (T); (3) T + 80 mg/L of sodium bentonite clay (CL; Astra-Ben-20, Prince Agri Products Inc., Quincy, IL); or (4) CL + 14 mg/L of SCFP (CL+SCFP; Diamond V, Cedar Rapids, IA). Ruminal fluid was collected 3 h after the morning feeding from 3 cannulated cows fed the same TMR, and rumen fluid from individual cows was used to prepare separate inocula. Each treatment was incubated in duplicate at 39°C for 0, 4, 8, 16, and 24 h in each of 3 runs. Adding T reduced total volatile fatty acid (VFA) concentration after 4 and 8 h and molar proportion of propionate after 4 and 24 h of incubation, relative to control. Adding sequestering agents (CL and CL+SCFP) with T did not affect total VFA concentration after 4 or 8 h, but increased total VFA after 16 h and tended to increase molar proportion of propionate after 24 h, compared with T. At 24 h, T had lower DM digestibility and higher NH-N concentration compared with the control. Thus, AFB1, even at very low concentration (0.75 µg/L), had detrimental effects on rumen fermentation and subsequently DM digestibility of the TMR. Adding sequestering agents did not prevent negative effects of T on rumen fermentation within 8 h of incubation; however, sequestering agents were effective after 16 h of incubation.