Social insects are prone to pathogen infection because of high exposure rates from social interactions. However, it remains unclear whether queens have enhanced pathogen resistance, since reproduction is largely confined to queens. Here, we used a natural host-pathogen system, the subterranean termite Reticulitermes chinensis and the entomopathogenic fungus Metarhizium anisopliae, to investigate the differences in allo-grooming, locomotion, and immune gene expression between queens and workers against pathogen infection. We found that fungal infection significantly reduced survival in both queens and workers. Infected queens received significantly more grooming time from sanitary nestmates than infected workers, but they returned much less grooming time to sanitary nestmates than infected workers. Infection resulted in a reduction in the average locomotion speed and distance of queens but had no effect on worker locomotion. Infection resulted in upregulated expression of two immune genes (termicin and transferrin), two antioxidant genes (CAT and SOD), and phosphate genes CYP450 in queens but not in workers. Our results indicated that eusocial termites evolved strategies that prioritize the reproductive castes welfare in defending against the pathogen infection to ensure continued reproduction and colony persistence.