Betaine, a trimethylglycine, is a natural ingredient of wheat and sugar beets. In the body, choline oxidation can lead to the formation of betaine. Betaine acts as an organic osmolyte and a methyl donor. It may also be fermented in the rumen. The objective of this experiment was to determine the effect of supplemental betaine on ruminal fermentation in dairy cows. The experiment included 3 rumen-cannulated Holstein dairy cows in a 3 × 3 Latin square design with 3 periods, 8 d each (1 d for in situ bags introduction and rumen fluid collection, 7 d wash-out), and 3 treatments of betaine at 0, 100, and 200 g placed into each Dacron bag and double sealed. Rumen fluid was collected and large mesh bags containing Dacron bags were introduced into the rumen at different time points (0, 1, 1.5, 2, 3, 6, 12, and 24 h). At each time point, 3 bags of each treatment were prepared. Dry and organic matter degradation was determined on dried, post in situ sample bags. Rumen fluid samples from different time points were analyzed for volatile fatty acid profile using gas chromatography. Data were analyzed using the Proc Mixed of SAS with significance declared at P ≤ 0.05. Organic matter degraded greater (P < 0.0001) when betaine retention time was longer than 12 h. There were increases in the ratio of acetate to propionate with betaine supplementation (1.7, 1.9, and 1.8 ± 0.03, for 0, 100, and 200 g betaine, respectively; P < 0.0001) and molar proportion of isovalerate with betaine supplementation (P < 0.005). Significant decreases in pH (6.4, 6.3, and 6.2 for 0, 100, and 200 g betaine, respectively) and molar proportion of propionate, butyrate, and isobutyrate with betaine supplementation were observed (P < 0.001 for all).There were no detectable changes in the molar proportion of acetate (0.42, 0.43, and 0.42 ± 0.006) or valerate (0.05, 0.04, and 0.05 ± 0.005). Overall, changes in volatile fatty acid profile observed suggest the possibility of an effect of betaine supplementation on rumen microbial populations, which warrants further investigation.