Nosema ceranae is a common and highly contagious fungal pathogen that primarily infects the gut of adult honeybees, causing nosemosis. As a chronic disease of the digestive system, it poses a global threat to honeybee health and colony sustainability. This study aimed to investigate the inhibitory effects of different concentrations of Scutellaria baicalensis aqueous extract on N. ceranae in the intestines of infected Apis cerana through feeding experiments. In addition, the therapeutic efficacy of its major active component, baicalin, was evaluated, and its potential molecular mechanisms of action were explored. The results showed that, compared with the control group, administration of S. baicalensis aqueous extract at concentrations of 1 mg/mL, 5 mg/mL, and 10 mg/mL significantly reduced midgut spore loads (p < 0.05). Further experiments showed that a 0.5 mg/mL baicalin sucrose solution, prepared with 0.5% (v/v) DMSO as co-solvent, exhibited optimal solubility and significantly inhibited the proliferation of spores in the honeybee midgut. Transcriptomic analysis of A. cerana revealed varying numbers of significantly differentially expressed genes among the baicalin-treated (HG) group, the co-solvent control (DMSO) group, and the blank control (C) group. Four candidate DEGs associated with the effects of baicalin were further identified, namely LOC108003965, LOC108000905, LOC107996681, and CYP4G11. Gene Ontology enrichment analysis showed that, in the comparison between the HG group and the C group, these DEGs were significantly enriched in six functional categories: iron ion binding, phosphoric ester hydrolase activity, heme binding, tetrapyrrole binding, hydrolase activity (acting on ester bonds), and oxidoreductase activity (acting on paired donors, with incorporation or reduction of molecular oxygen). Collectively, these results demonstrate that S. baicalensis aqueous extract effectively inhibits the proliferation of N. ceranae within the host, and its active component, baicalin, exhibits a similar inhibitory effect. The present study proposes a novel strategy in which baicalin may enhance host endogenous chitinase-related activity to target and disrupt the spore wall, offering a new perspective for the prevention and control of honeybee nosemosis.