The ban on the use of antibiotics as growth promoters in animal feeds in the European Union has stimulated research on potential alternatives. Recently, propyl-propane thiosulfonate (PTSO), a stable organosulfurate compound of garlic, was purified. The objectives of the current study were to investigate the potential effects of PTSO on rumen microbial fermentation and to define effective doses. Two experiments were conducted using dual-flow continuous culture fermenters in 2 replicated periods. Each experimental period consisted of 5 d for adaptation of the ruminal fluid and 3 d for sampling. Temperature (39°C), pH (6.4), and liquid (0.10 h(-1)) and solid (0.05 h(-1)) dilution rates were maintained constant. Samples were taken 2 h after feeding and from the 24-h effluent. Samples were analyzed for volatile fatty acids (VFA) and nitrogen fractions, and degradation of nutrients was calculated. In addition, 24-h effluents from experiment 2 were analyzed for their fatty acid (FA) profile. Treatments in experiment 1 included a negative control without additive, a positive control with monensin (12mg/L), and PTSO at 30 and 300mg/L. The addition of 30mg/L did not affect any of the measurements tested. The addition of 300mg/L reduced microbial fermentation, as suggested by the decreased total VFA concentration, true degradation of organic matter and acid detergent fiber, and a tendency to decrease neutral detergent fiber degradation. Experiment 2 was conducted to test increasing doses of PTSO (0, 50, 100, and 150mg/L) on rumen microbial fermentation. At 2 h postfeeding, total VFA and molar proportion of propionate responded quadratically, with higher values in the intermediate doses. Molar proportions of butyrate increased and branched-chain VFA decreased linearly as the dose of PTSO increased. In the 24-h effluents, total VFA, acetate, and branched-chain VFA concentrations decreased linearly and those of propionate responded cubically with the highest value at 100mg/L. Saturated FA decreased and unsaturated FA increased linearly with increasing dose of PTSO. The concentration of trans-10,cis-12 conjugated linoleic acid decreased by 78.5% with addition of PTSO at the highest dose (150mg/L). Results suggest the potential of PTSO to modify ruminal fermentation in a direction consistent with higher propionate molar proportion, higher outflow of unsaturated FA, and low trans-10,cis-12 conjugated linoleic acid in an effective dose between 50 and 100mg/L.