BACKGROUND: Microbial fermentation is regarded as the best bioimprovement technique for changing the physicochemical characteristics and structural makeup of carbohydrates. In the present study, lotus root starch (LRS) was co-fermented with Saccharomyces cerevisiae and Lactiplantibacillus plantarum. The effects of single- and mixed-strain fermentation on the structure, physicochemical properties and digestibility of starch were investigated at different fermentation times. RESULTS: The results showed that cracks and grooves appeared on the surface of fermented LRS, particularly with prolonged mixed fermentation. Additionally, the particle size, dissociation, freeze-thaw dehydration shrinkage and hydration characteristics of starch significantly decreased. Structural characterization showed that fermentation promoted the alignment of LRS long chains towards shorter sequence structures, enhanced hydrogen-bonding interactions and increased the relative crystallinity of LRS by degradation of the amorphous region. Mixed fermentation also increased the viscosity, springiness, thermal stability and shear stability of LRS gels compared to single fermentation, effectively improving the digestive performance. CONCLUSION: This study has confirmed that co-fermentation with L. plantarum and S. cerevisiae significantly influences both the multiscale structure and physicochemical properties of LRS. These results provide promising strategies for processing and nutritional enhancement of fermented lotus root products. © 2025 Society of Chemical Industry.