Ruminants are less susceptible to mycotoxins than monogastrics (Kurmanov, 1977). Rumen microbes are responsible for the metabolization of the toxins. The formed metabolites can be non-toxic, but also equally or even more toxic than the parent mycotoxins. Although often no acute symptoms can be observed, a long-term intake of a diet contaminated with fumonisins (FUM) can lead to reduced feed intake and a loss in milk production. A purified enzyme (fumonisin esterase, FumD) was developed which transforms fumonisin B1 (FB1) into hydrolyzed FB1 (HFB1) and 2 tricarballylic acid chains (TCA) (Hartinger and Moll, 2011). Remaining TCA and HFB1 are not toxic any more. Aim of the in vitro batch trial was to demonstrate that FumD is efficient in biotransforming FUM into non-toxic metabolites in rumen fluid. Rumen content was taken immediately after slaughter and transported to the lab. The rumen content was incubated under anaerobic conditions together with feed for 24 h in batch reactors (100 mL containers), starting at a pH of 6.5. The 15 reactors were divided into 3 treatment groups: group 1: negative control, group 2: FUM (50 mg/kg feed) and group 3: FUM + FumD (100 Units/kg feed). Samples were taken after 10 min, 1 and 24 h of incubation and were analyzed for FB1 and HFB1 content via HPLC-MS/MS. Statistical analysis was performed using SPSS 19.0. Normal distribution was tested and for comparison of groups One-Way ANOVA or non-parametric Kruskal Wallis test was performed. Within the incubation period of 24 h, it was shown that FB1 was slowly released from FUM culture material into the fermentation broth (FB1 13.4 µmol/L). However, there was no hydrolysis of FB1 to HFB1 observed in group 2 (HFB1 0 µmol/L). In group 3 FB1 was completely converted into the non-toxic hydrolyzed form HFB1 within 10 min (7.3 µmol/L). Moreover, between 1 and 24 h, the enzyme completely hydrolyzed slowly released FB1 into HFB1 (HFB1 9.6 µmol/L). Under field conditions the fumonisin contamination of feed used for farm animals is on average less than 5 ppm, which is 10 times lower than the concentration used in this experiment. Under in vitro conditions, FumD was capable of completely degrading FB1 within rumen environment and remained active between 1 and 24 h.