Glycogen synthase kinase-3β (GSK-3β) is a ubiquitously expressed serine/threonine kinase that plays a pivotal role in regulating innate and adaptive immunity by modulating the production of pro- and anti-inflammatory cytokines. To investigate specific regulators of GSK-3β in innate immunity, we developed small molecule inhibitors that are tightly interact with three ATP-binding sites of GSK-3β through a systematic Fragment-based de novo design (FBDND). Among the leading candidates, G3I was found to modulate early macrophage polarization by downregulating the expression of mRNAs of M2-like markers such as CD206, Arg-1, CCL18 and upregulating the markers for M1-like macrophages such as CD86 and CCR7. Additionally, G3I-treated M1-like macrophages demonstrated enhanced pro-phagocytic and cytotoxic tendency against tumor cells, and M2-like macrophages exhibited altered migration behavior. G3I significantly decreased NF-κB activity and influenced the production of inducible nitric oxide synthase (iNOS) and pro-inflammatory cytokines such as TNF-α and IL-6 through Toll-like receptor 4 (TLR4) signaling stimulated by lipopolysaccharide (LPS). Furthermore, G3I-treated M1 macrophages inhibit tumor cell proliferation and enhance macrophage phagocytic activity as well as M2 macrophages decrease tumor migration. These findings suggest that G3I regulates the balance of macrophage polarization in the tumor microenvironment, and enhances the phagocytic and microbicidal activities of macrophages, indicating its potential as a novel therapeutic application in cancer treatment offering a potential breakthrough in targeting immune modulation and improving anti-tumor responses.