Anti-double-stranded DNA (dsDNA) IgG causes renal damage in patients with lupus nephritis by cross-reacting with multiple autoantigens, including alpha-actinin-4, in mesangial cells (MCs). However, how the cross-reactions play a role in mesangial phenotypic abnormalities is not well understood. Here, we investigated the effects of the fragment antigen-binding (Fab) of anti-dsDNA IgG3 on the biochemical properties of alpha-actinin-4. Experiments revealed that anti-dsDNA Fab specifically binds to alpha-actinin-4, but not G-actin. The binding by anti-dsDNA Fab sequentially increases the positive charge of alpha-actinin-4 and inhibits the affinity of alpha-actinin-4 to calcium ions. By the low shear viscosity and a co-sedimentation assay, we found that the alpha-actinin-4-induced F-actin gelation improves when anti-dsDNA Fab is added. However, the Fab control has no such effect on F-actin gelation. Furthermore, the in vitro cultured MCs exhibit higher F-actin expression and transforming growth factor-β1 synthesis after the incubation with anti-dsDNA Fab. Therefore, our results indicated that anti-dsDNA Fab may enhance F-actin formation by the proprietary modification of alpha-actinin-4, which could partially explain the myofibroblast-like phenotype of MCs in anti-dsDNA-positive lupus nephritis.