〔Abstract〕 To explore the role and molecular mechanism of parthenolide (PTL) in regulating tumor- associated macrophage (TAM) polarization to inhibit the malignant progression of gallbladder cancer cells. Methods M0 macrophages were co-cultured with human gallbladder cancer cell line GBC-SD and treated with PTL, and the cells were divided into control group, TAM group, TAM+PTL group. Flow cytometry was used to determine the levels of CD86 and CD206 in macrophages. RT-qPCR and Western blot were used to determine the expressions of CD163, interleukin-10 (IL-10) and arginase-1 (Arg-1). ELISA and Western blot were used to determine the secretion of CC chemokine ligand 2 (CCL2) in the supernatant of GBC-SD cells and the expression of CCL2 in the cells. GBC-SD cells were transfected with a CCL2 overexpression vector and divided into TAM, TAM+PTL, TAM+PTL+OE-NC, TAM+PTL+OE-CCL2 groups. Flow cytometry was used to assess CD86 and CD206 levels in macrophages, RT-qPCR and Western blot were used to assess CD163, IL-10 and Arg-1 expression. CCK-8 assay, plate-based colony formation assay and Transwell chamber assay were used to assess GBC-SD cell proliferation, migration and invasion, respectively. Results Compared with the TAM group, the TAM+PTL group showed increased CD86 expression and decreased CD206 expression in macrophages, the mRNA and protein expressions of CD163, IL-10 and Arg-1 were downregulated, the CCL2 content and protein expression in GBC- SD cells decreased, cell proliferation activity, colony formation, migration and invasion were all reduced (P<0.05). Compared with the TAM+PTL group, the TAM+PTL+OE-CCL2 group showed decreased CD86 expression and increased CD206 expression in macrophages, the mRNA and protein expressions of CD163, IL-10 and Arg-1 were upregulated. Cell proliferation activity was enhanced, and colony formation, migration, and invasion all increased (P<0.05). Conclusion Parthenolide inhibits the polarization of macrophages in TAMs to M2 type by downregulating CCL2 expression in gallbladder cancer cells, thereby preventing the proliferation, migration and invasion of gallbladder cancer cells.