Background: miRNA has been implicated in regulating cholesterol homeostasis, a critical factor in gallstone formation. Here, we focused on elucidating the role of miR-146a in this pathological process. Methods: C57BL/6 mice were fed with lithogenic diet (LD) and injected with miR-146 antagomir (anta-146) via the tail vein for various weeks. The gallbladders and liver tissues were collected for cholesterol crystal imaging, gallstone mass quantification, and molecular analysis. Levels of cholesterol, bile salt, phospholipids, and metabolic parameters in serum and bile were assessed by ELISA. A 3 ' UTR reporter gene assay was used to verify the direct target genes for miR-146. The relative expression of metabolism genes was analyzed by quantitative real-time PCR and immunoblotting. Results: miR-146a-5p expression was reduced in mice and clinical samples with gallstones. Anta146 treatment effectively prevented LD-induced gallstone formation in mice without hepatic and cholecystic damage. The mice treated with anta-146 exhibited beneficial alterations in bile cholesterol and bile acids and lipid levels in the blood. A key biliary cholesterol transporterMegalin was identified as a direct target of miR-146. Anta-146 administration upregulated megalin expression, thereby ameliorating impaired gallbladder cholesterol absorption associated with the LXR-megalin/cubilin pathway. Conclusion: The data demonstrates that miR-146 modulates gallbladder cholesterol absorption by targeting megalin, and prevents the pathogenesis of cholesterol gallstones.