The minimum effective dose of the Mycoplasma synoviae-H (MS-H) vaccine was determined through protection against experimental challenge. Chickens were vaccinated by eyedrop with the following doses of a vaccine: 1.2 × 10⁵, 2.4 × 10⁵, 4.8 × 10⁵, 9.6 × 10⁵, 1.92 × 10⁶, and 3.84 × 10⁶ color change units (CCU), then challenged 6 wk after vaccination. Rapid serum agglutination results indicated that 100% of birds receiving an MS-H dose of ≥4.8 × 10⁵ CCU had antibodies to MS and enzyme-linked immunosorbent assay results showed that 60% of birds receiving a dose of 4.8 × 10⁵ or 9.6 × 10⁵ CCU and 100% of birds receiving a dose of 1.92 × 10⁶ or 3.84 × 10⁶ had antibodies to MS. At postmortem after challenge, the following parameters were significantly lower in birds vaccinated with an MS-H dose of ≥4.8 × 10⁵ CCU: air sac (AS) lesion severity; incidence of AS lesions; mucosal thicknesses in the upper trachea, middle trachea, and lower trachea (LT); and MS colonization of the LT and AS. It was concluded that an MS-H dose of 4.8 × 10⁵ CCU was sufficient to elicit an antibody response in birds, prevent MS colonization in the LT and AS, and protect against AS lesions caused by an experimental MS and infectious bronchitis virus challenge.