Background <p>It is generally known that kidney stone disease (KSD) is associated with alterations in urinary microbiome, but the roles of the urinary microbiome in KSD pathogenesis remain unclear.</p> Methods <p>This study addressed the impact of <i>Lactobacillus acidophilus</i> (a commensal bacterium found in normal urine) on renal epithelial integrity, calcium oxalate monohydrate (COM) crystal-cell adhesion, expression of membrane receptors of COM crystals, and oxalate degradation under oxalate-induced stress (a known inducer of KSD). Inner medullary collecting duct cells (mIMCD-3) were cultured for 24 h under control or oxalate-induced (by 0.6 mM sodium oxalate; NaOx) conditions without or with <i>L. acidophilus</i> (at 1 × 10³ colony-forming unit (CFU)/ml) co-incubation.</p> Results <p>NaOx reduced transepithelial resistance (TER) of the mIMCD-3 monolayer and downregulated ZO-1, a tight junction (TJ) protein. Additionally, NaOx enhanced the COM crystal-binding capability of mIMCD-3 cells by upregulating a COM crystal receptor, annexin A2, on cell membranes. Such harmful effects of NaOx were abolished when mIMCD-3 cells were co-cultured with <i>L. acidophilus</i>. Moreover, culturing <i>L. acidophilus</i> in artificial urine (AU) supplemented with NaOx for 24 h revealed that the oxalate level in AU decreased, suggesting the oxalate-degrading activity of the bacterium in an AU environment.</p> Conclusions <p><i>L. acidophilus</i> prevented oxalate-mediated renal epithelial barrier disruption and COM crystal adhesion to renal epithelial cells by preserving ZO-1 and annexin A2 expression at their basal levels, at least in part, via its oxalate-degrading property.</p> Clinical trial number <p>Not applicable (This is not a clinical trial).</p>