The recalcitrance of cellulosic biomass greatly hinders its enzymatic degradation. Expansins induce cell wall loosening and promote efficient cellulose utilization; however, the molecular mechanism underlying their action is not well understood. In this study, TlEXLX1, a fungal expansin from Talaromyces leycettanus JCM12802, was characterized in terms of phylogeny, synergy, structure, and mechanism of action. TlEXLX1 displayed varying degrees of synergism with commercial cellulase in the pretreatment of corn straw and filter paper. TlEXLX1 binds to cellulose via domain 2, mediated by CH–π interactions with residues Tyr291, Trp292, and Tyr327. Residues Asp237, Glu238, and Asp248 in domain 1 form hydrogen bonds with glucose units and break the inherent hydrogen bonding within the cellulose matrix. This study identified the expansin amino acid residues crucial for cellulose binding, and elucidated the structure and function of expansins in cell wall networks; this has potential applications in biomass utilization.