<p>In inflammation, osteopontin (OPN) acts as both a stone matrix component for calcium oxalate (CaOx) crystal formation and an inflammatory mediator. While previous studies have demonstrated the individual roles of OPN and macrophages (Mφ) in renal CaOx stone formation during inflammation, their interaction remains poorly understood. This study aimed to elucidate the role of OPN in modulating Mφ function during crystal formation, using an ex vivo model. Bone marrow-derived macrophages (BMDM) were isolated from eight-week-old male C57BL/6J wild-type and OPN knockout mice. BMDMs from OPN-positive (BMDM^OPN+) and OPN-negative (BMDM^OPN−) mice were co-cultured with fluorescently labeled CaOx monohydrate (COM) crystals for phagocytosis assays and analyzed using the IN Cell Analyzer 6000. We further performed real-time quantitative reverse transcription PCR and RNA sequencing to identify gene expression profiles and clarify the role of OPN in Mφ function. The assay analysis demonstrated that phagocytosis rates were significantly higher in BMDM^OPN− than in BMDM^OPN+. Inflammatory markers, such as IL-6, TNF, CD44, were upregulated following COM exposure, and IL-6 expression was significantly lower in BMDM^OPN− than in BMDM^OPN+. RNA sequencing revealed that BMDM^OPN− exhibited a less pro-inflammatory and more anti-inflammatory phenotype (<i>Csf2</i>^<i>l</i>ow, <i>Irf5</i>^low, <i>Itgax</i>^low, <i>Csf1</i>^high, <i>Cd163</i>^high), resembling M2-like Mφs. Further functional analysis indicated that OPN knockdown in Mφs increased the S100 family and CREB signaling, which enhanced the M2-like phenotype shift and phagosome formation. In conclusion, OPN plays a critical role in enhancing pro-inflammatory Mφ function, potentially limiting COM phagocytosis. Modulating OPN expression in circulating Mφs may represent a therapeutic approach for kidney stone disease.</p>