<p><i>Mycoplasma bovis</i> (<i>M. bovis</i>) is the primary pathogen causing subclinical mastitis (SCM) in dairy cows. Although vaccination is a cornerstone of disease control, its efficacy as a therapeutic intervention in naturally infected SCM cases remains largely unexplored. This study is the first to evaluate the immunological intervention effectiveness of an <i>M. bovis</i>-BoAHV-1 combined vaccine in a field-based model of <i>M. bovis</i> natural infection. The vaccine was administered through the subcutaneous (SC) or intranasal (IN) route, with efficacy assessed using positive control and blank control groups as references. The results indicated that the combined vaccine effectively shortened the duration of SCM and alleviated the symptoms, with the SC route demonstrating superior intervention effects compared to the IN route. The pathogen excretion in the SC group decreased significantly and ceased by day 52. Meanwhile, somatic cell counts declined rapidly, with all cows achieving mastitis-negative status by day 49. The pathogen shedding duration and abnormal somatic cells status in the SC group were shortened by 7 and 14 days, respectively, compared to the IN group. The recovery process was further evidenced by the normalization of key mammary health biomarkers, coupled with a notable restoration of lactation performance, confirming the clinical resolution of the infection. Mechanistically, the combined vaccine markedly enhanced <i>M. bovis</i>-specific lymphocyte proliferation capacity, indicating effective stimulation of antigen-specific immunity. These findings provide the first field-practice confirmation of the therapeutic intervention efficacy of a combined vaccine against SCM, offering a promising and non-antibiotic solution to address antibiotic-resistant mastitis.</p>