ABSTRACT The purpose of the present study was to evaluate effects of medium-chain fatty acid-cyclodextrin (CD) complexes on ruminal methane and volatile fatty acid production, and protozoal activity in vitro. Medium-chain fatty acid-CDs used in this study were caprylic acid (C8)-αCD or -βCD, capric acid (C10)-αCD or -βCD, and lauric acid (C12)-αCD or -βCD. A 60-mL of diluted rumen fluid was incubated anaerobically at 38°C for 6 h with the addition of the complex (10–40 mg as fatty acid). Each of the fatty acid-CDs reduced the number of protozoa, with the order C10 > C12 > C8, and βCD complexes were more effective than αCD complexes. Molar proportions of acetic acid remained unchanged with the addition of fatty acid-CD, while that of propionic acid increased, being significant for C8-αCD and βCD, and C10-αCD and βCD (P < 0.05). Hydrogen production decreased by about 70% of control with the addition of 40 mg of C8 and C10-CD, on the other hand, it tended to increase with the addition of C12-CD in both αCD and βCD. Methane production decreased by about 20% with the addition of 40 mg of complexes, except for C10-βCD, which significantly reduced methane production by about 60%. In conclusion, the addition of C8 or C10-CD to ruminant diets may be effective in reducing methane production.