Zearalenone (ZEN) is one of the most frequently detected mycotoxins in cereals and feed all over the world. Among farm animals, it is thought that ruminants are less sensitive to ZEN. However, the α-ZEL metabolite produced in the rumen has higher estrogenic potential than ZEN. There is evidence that the ZEN degrading enzyme zearalenone hydrolyase (ZenA) exhibits activity against ZEN by forming the non-estrogenic hydrolyzed zearalenone (HZEN) as the primary reaction product. In this study, we evaluated the effect of ZenA as a feed additive on ZEN kinetics in the rumen.In a 10-d feeding trial, 4 rumen-cannulated Holstein Friesian cows (nonlactating, non-gestating) were used to assess the kinetics of ZEN, depending on the experimental day, all animals received either (1) a corn portion containing 5mg of ZEN, (2) a corn portion containing 5mg of ZEN supplemented with ZenA, or (3) a uncontaminated corn portion(control). Rumen fluid and fecal samples were taken each day at various time points and ZEN, α-ZEL, and HZEN concentrations were determined using high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) analysis. For statistical analysis, a t-test was performed if requirements of normal distribution and homogeneity of variances were met. Otherwise, a non-parametric test (Mann-Whitney U test) was used. When animals received ZEN contaminated feed, ZEN, α-ZEL and HZEN was detected in the rumen fluid at a concentration of 25.04 nmol/L, 2.42 nmol/L and < LOQ, respectively. When animals received ZEN contaminated feed supplemented with ZenA, ZEN, α-ZEL and HZEN was detected in the rumen fluid at a concentration of 2.18 nmol/L, < LOQ and 41.75 nmol/L, respectively. Detected ZEN (P = 0.001) and α-ZEL (P < 0.001) levels were significantly lower and HZEN (P < 0.001) levels significantly higher after administration of ZenA. Fecal samples showed the same significant (P < 0.01) shift from the estrogenically active ZEN and α-ZEL to the non-toxic HZEN. We conclude that ZenA applied as a feed additive was effective in transforming ZEN to HZEN in the rumen of dairy cows and it is therefore, a promising approach to prevent toxic effects of ZEN contaminated feed.