The demand for silk increases as its economic value rises. Silk production by the silkworm, Bombyx mori L., is linked to the quality of the silkworm diet, leading to efforts to nutritionally enrich the mulberry (Morus alba L.) diet of silkworm larvae. Previous studies have established that spermidine, a polyamine, enhances larval growth in B. mori, subsequently increasing silk production. However, its role in improving the nutritional quality of the silkworm diet is not known. In this study, we evaluated the effects of spermidine-treated diet on the nutritional indices, polyamine levels, and antioxidant potential in fifth-instar larvae. We also assessed the effect of consumption of the spermidine-treated diet on the larval gut microbiome, which impacts digestion and assimilation of nutrients. Larvae consuming the spermidine-treated diet showed a significant increase in the efficiency of conversion of ingested food and digested food, intracellular polyamine levels (especially the conjugated and free fraction), antioxidant potential and cell viability, and both diversity and number of bacterial communities. These findings suggest that feeding mulberry leaves fortified with spermidine enhances nutritional efficiency in the B. mori larvae and may represent a method of increasing silk production by B. mori.