Fumonisin (FMN) is a mycotoxin produced by Fusarium species and has been found in feeds fed to dairy cattle. The effects of FMN on ruminal bacterial growth and fermentation were measured with batch culture. Experimental feeds contained 0, 100 or 200 ppm FMN and were arranged as a completely randomized design (CRD). All feeds were incubated at 39°C with buffer and strained ruminal fluid. Culture optical density (OD) was measured to estimate microbial populations. The cultures were sampled at 0, 6, 12, 24 and 36 h to measure the fermentation end products, pH and OD. OD increased over time with no differences among treatments except at 12 h. At 12 h, OD was higher for cultures fed diets containing FMN at 100 (1.66) and 200 (1.62) ppm versus feed without FMN (1.41). Culture pH decreased over time and was not significantly different due to treatments over the entire study. Total concentration of VFA and concentrations of acetic acid, propionic acid, and butyric acid increased over time with no differences among treatments. Feed containing no FMN supported higher concentrations of ammonia and branch chain VFA (BCVFA) versus the other treatments from 6 h through 36 h. There was a high correlation between ammonia and BCVFA concentrations at sample times (r = 0.97). The results indicated that FMN changed ruminal microbial fermentation of protein as measured by changes in BCVFA and ammonia in this in vitro fermentation system. The effect of these changes in rumen fermentation on animal performance should be investigated.