The aim of the present study was to evaluate the effects of Lactobacillus rhamnosus GG (LGG; ATCC 53013) on growth performance and hepatotoxicity in calves fed a single dose of aflatoxin B 1 (AFB 1) and to investigate the absorption, distribution, and elimination of AFB 1 and the hydroxylated metabolite aflatoxin M 1 (AFM 1) in rumen fluid, blood, and excretions. Twentyfour male Holstein calves were blocked for body weight and age and were randomly assigned to 1 of 3 treatment groups: (1) untreated control, (2) treated with 4.80 mg of AFB 1 (AFB 1 only), or (3) treated with 1 × 10 10 cfu of LGG suspension and 4.80 mg of AFB 1 (AFB 1 plus LGG). The calves received LGG suspension in 50 mL of phosphate-buffered saline daily via oral administration for 14 d before and on the day they received a single oral dose of AFB 1. Body weight was recorded at the beginning of the study (before LGG administration), at the day of AFB 1 administration, and at the end of the trial. Rumen fluid, blood, urine, and feces samples were collected continuously for 96 h after AFB 1 administration. Average daily gain (ADG) and plasma biochemical parameters were analyzed, and concentrations of AFB 1 and AFM 1 in the samples were determined for monitoring excretion pattern and toxicokinetics. The results showed that ADG was lower in AFB 1-treated animals; LGG administration partially mitigated the decrease in ADG (0.85 ± 0.08 vs. 0.76 ± 0.18 kg of gain/d). The AFB 1 treatment increased plasma aspartate aminotransferase, alkaline phosphatase, and lactate dehydrogenase levels. Administration of LGG alleviated the AFB 1-induced increase in plasma enzymes activity. The excretion patterns of AFB 1 and AFM 1 were surprisingly regular; toxins were rapidly detected in all samples after a single oral dose of AFB 1 , and the peak of toxins concentrations was sequentially reached in rumen fluid, plasma, urine, and feces (except AFM 1 in rumen fluid), followed by an exponential decrease. The excretion curves showed that AFB 1 and AFM 1 concentrations were the highest in feces and urine, respectively. Administration of LGG decreased the concentrations of free AFB 1 and AFM 1 in rumen fluid and reduced the release of toxins into plasma and urine. Toxicokinetic parameters (except for the time of maximum concentration and the terminal half-life) were reduced by LGG administration. In conclusion, the absorption, distribution, and excretion of AFB 1 and AFM 1 were rapid in calves fed a single dose of AFB 1. Urine was the main route for the excretion of AFM 1 , and the clearance pattern from the peak of concentration was well fitted by exponential decreasing function. Administration of LGG reduced the absorption of AFB 1 in the gastrointestinal tract by increasing the excretion via the feces, thus alleviating the hepatotoxic effect of AFB 1 .