Abstract <p>This study, for the first time, applied glucose—a cost-effective, non-aromatic co-metabolite—to the co-metabolism of pyrene, a polycyclic aromatic hydrocarbon (PAHs). The effects of glucose on the co-metabolic degradation of pyrene by <i>Stenotrophomonas rhizophila</i>, a PAHs-degrading bacterium, and its potential mechanisms were investigated. The results demonstrated that co-metabolism reduced the biodegradation half-life by 18 days and increased degradation efficiency from 21.02% to 29.22% (P&lt;0.01), with 200 mg/L glucose identified as the optimal concentration. Co-metabolism significantly affected the microbial surface characteristics and the pollutant adsorption. Specifically, microbial cell length exhibited a significant increase from 0.77 ± 0.18 μm to 0.88 ± 0.15 μm, accompanied by a vertical height increment of 5.24 nm. Notably, co-metabolism effectively restored structural integrity to damaged surface areas, which consequently generated novel binding domains for enhanced pollutant adsorption. Moreover, co-metabolism enhanced the synthesis of microbial surface substances and induced the appearance of -SH groups in glutathione, promoting normal immune function. Microbial surfaces in the co-metabolic medium exhibited higher hydrophobicity and a neutral Zeta potential. Co-metabolism also enhanced microbial metabolic activity, increasing the activities of enzymes involved in degradation. Notably, it increased the activity of key degradation-associated enzymes, particularly catechol 2,3-dioxygenase, facilitating the meta-cleavage pathway of catechol and reducing overall toxicity of mixed metabolites during biodegradation, as predicted by ECOSAR software. These findings underscore the potential of co-metabolism in the bioremediation of PAHs.</p> Graphical Abstract <p></p>