D-mannose, a naturally occurring monosaccharide, is commonly utilized in the management of feline urinary tract disease, yet its immunomodulatory effects and influence on the gut microbiota in cats remain unclear. This study investigated the effects of D-mannose supplementation on immune function, hematological and biochemical indices, and intestinal microbiota composition in adult cats. Eighteen healthy British Shorthair cats were randomly assigned to a control group or a D-mannose group, receiving daily oral supplementation with d-glucose or D-mannose (0.5 g/kg), respectively, for a period of 14 days. Administration of D-mannose resulted in elevated serum IgG levels and improved hematological parameters associated with oxygen transport (HGB, MCV, MCH, and RDW-SD) and coagulation function (MPV, PDW, and PCT) (P ≤ 0.05). 16S rRNA gene sequencing revealed that the gut microbial alpha diversity remained stable, whereas beta diversity exhibited significant variation (P < 0.001). Moreover, D-mannose enhanced microbial homeostasis, as reflected by an elevated gut microbiota health index (GMHI) and a reduced microbial dysbiosis index (MDI) (P < 0.01). Taxonomic analysis showed enrichment of Coprococcus sp. AF21-14LB (P < 0.001) and reduced abundance of Peptacetobacter hiranonis and Lactimicrobium massiliense (P < 0.05). In conclusion, D-mannose supplementation contributes to enhanced systemic immunity and improved hematological parameters, while fostering gut microbial homeostasis through modulation of bacterial taxa and community structure, supporting its potential as a functional dietary additive for feline health.