Background <p><i>Acanthamoeba</i> spp., which are free-living protozoan parasites, are etiological agents for <i>Acanthamoeba</i> keratitis and granulomatous amoebic encephalitis. Macrophages participate in the host defense response to resist <i>Acanthamoeba</i> spp. This study examined the effect of <i>Acanthamoeba castellanii</i> cysteine protease 3 (<i>Ac</i>CP3) on macrophage activation during inflammatory responses and explored the underlying mechanisms.</p> Methods <p>The effects of recombinant <i>Ac</i>CP3 (r<i>Ac</i>-CP3) stimulation on inflammatory factor levels and macrophage polarization were examined using murine macrophage cells (RAW264.7 cells). Western blotting assay was carried out for analyzing TLR4/NF‑κB pathway-related protein levels. In addition, phosphorylated NF-κB was examined for its nuclear transport using immunofluorescence. The effect of the NF-κB inhibitor pyrrolidinedithiocarbamate ammonium (PDTC) on r<i>Ac</i>-CP3-induced M1 polarization was analyzed. Furthermore, RAW264.7 cells were co-cultivated using <i>Ac</i>CP3 knockdown trophozoites to examine indicators of M1 polarization and pathway-related protein levels.</p> Results <p>As revealed by quantitative real-time polymerase chain reaction (qRT-PCR), western blotting, and enzyme-linked immunosorbent assays, treatment with r<i>Ac</i>-CP3 upregulated the mRNA, protein, and secretion levels, respectively, of Il6, Il1b, Tnfa, and Ifng in macrophages. Flow cytometric analysis demonstrated that r<i>Ac</i>-CP3 promoted Cd86⁺ cell (macrophage) proliferation. Additionally, r<i>Ac</i>-CP3 upregulated Nos2 expression and nitric oxide (NO) production, indicating that r<i>Ac</i>-CP3 promotes macrophage polarization toward an M1-like phenotype. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis demonstrated that the NF-κB pathway was among the top 20 significantly enriched pathways. Treatment with r<i>Ac</i>-CP3 upregulated the levels of Tlr4, p-Rela, and p-Nfkbia in RAW264.7 cells. Immunofluorescence analysis demonstrated the nuclear translocation of p-Rela. Pretreatment with the NF-κB inhibitor PDTC downregulated the Tlr4, p-Rela, and p-Nfkbia levels in r<i>Ac</i>-CP3-treated cells. Additionally, PDTC significantly mitigated the r<i>Ac</i>-CP3-induced upregulation of Nos2, NO and pro-inflammatory factor production. <i>Ac</i>CP3 knockdown decreased the number of Cd86⁺ cells and suppressed <i>Acanthamoeba</i> trophozoite-induced Nos2 upregulation and NO production. Additionally, <i>Ac</i>CP3 knockdown downregulated Tlr4, p-Rela, and p-Nfkbia in RAW264.7 cells. PDTC and <i>Ac</i>CP3 knockdown suppressed the r<i>Ac</i>-CP3-induced M1 macrophage polarization.</p> Conclusions <p><i>Ac</i>CP3 promotes M1 macrophage polarization through the TLR4/NF-κB pathway and may exacerbate inflammation through upregulating pro-inflammatory cytokines.</p> Graphical Abstract <p></p>