As a glucose production method, the depolymerization of cellulose into glucose could be the one of the practical methods for sustainable bioproduction. However, cellulose’s crystalline structure and extensive inter- and intramolecular hydrogen bonding limit efficient enzymatic hydrolysis. In this study, we explored the heterologous expression of loosenin-like proteins from Phanerochaete carnosa in Escherichia coli as auxiliary proteins to enhance cellulase-mediated cellulose depolymerization. Four loosenin homologs (LOOL2, LOOL7, LOOL9 and LOOL12) were codon-optimized, cloned, and expressed in multiple E. coli strains. Of the four loosening proteins, LOOL2 and LOOL12 were only expressed in soluble form when chaperone proteins were present. Purified recombinant proteins were characterized for solubility, purity, and their capacity to modify cellulose surface morphology and synergy with cellulase. Surface alterations of Avicel were assessed by scanning electron microscopy after incubation with functionally expressed loosenins. Optimal conditions for enhanced hydrolysis were determined by measuring cellulase activity in the presence of varying LOOL concentrations at pH 5.0 and 50 °C. Our results indicate that recombinant loosenins can be expressed in soluble form in E. coli and that loosenins can influence cellulose surface accessibility, contributing to increased cellulase efficiency under selected conditions. The study provides a foundational framework for leveraging loosenin-assisted cellulose hydrolysis, addressing the critical need for sustainable glucose sources in biotransformation and fermentation processes. Further studies on the loosenin variants or enzyme fusion would be beneficial to maximize practical yields for industrial applications.