TGF-β isoforms play crucial roles in diverse cellular processes. Therefore, targeting and inhibiting TGF-β signaling pathway provides a potential therapeutic opportunity. TGF-β isoforms bind and bring the receptors (TβRII and TβRI) together to form a signaling complex in an ordered manner.Herein, an antagonistic variant of TGF-β (AnTβ) has been designed and prepared to inhibit the formation of signaling complex and consequently its signaling pathway. This TGF-β homodimeric variant contains intact TβRII binding sites and blocked TβRI binding sites by substituting three peptide segments. So, AnTβ could only bind to TβRII, but prevent binding and recruitment of TβRI to form a signaling complex.A reliable model of AnTβ was built and refined using molecular dynamics (MD) simulation, followed by investigating the interactions of AnTβ with the receptors using in silico docking studies. After expression of disulfide-linked AnTβ in a SHuffle strain and purification of the protein using affinity chromatography, its biological activity was evaluated using Mink lung epithelial cells (Mvl Lu).No meaningful significant changes in AnTβ structure were observed when compared with the native protein. Based on the docking analysis, AnTβ binds to TβRII similar to TGF-β and its binding to TβRI was diminished considerably which was consistent with our design purpose. Cell-based bioassay indicated that AnTβ could modulate TGF-β-induced cell growth inhibition.Our analysis suggests that the antagonistic potency of AnTβ can be used as an anti-TGFβ signaling factor in the future perspectives.