Immune-mediated tissue destruction of graft-vs-host disease (GvHD) remains a major barrier to greater use of bone marrow transplantation (BMT). It is found that alloreactive donor-derived T cells activated through their T cell receptor (TCR) are primarily the major contributors to the immunopathobiology of GvHD. Protein kinase C-theta (PKCtheta), a crucial, early downstream kinase of TCR signaling, enhances T cell activation, thereby promoting alloreactive responses such as differentiation, proliferation, migration, and cytotoxicity. Thus, delineating specific ways of interfering PKCtheta signaling is beneficial for the GvHD treatment or prevention. Here, we investigated the molecular mechanisms driven by PKCtheta in T cells by establishing preclinical all-murine and humanized GvHD mouse models as well as in vitro primary cell cultures. We found that both CD4 and CD8 T cells expressing high levels of activated PKCtheta contributed to the development of GvHD. Genetic deletion or chemical inhibition of PKCtheta prevented the GvHD induction via preventing expression of proinflammatory cytokines. In addition, we established an effective strategy for intracellular antibody delivery against activated PKCtheta by cell-penetrating peptide mimics (CPPMs). CPPM: Antibody (cell-penetrating antibody) complexes were readily introduced with high efficacy into hard-to-transfect T cells, dampening PKCtheta-specific downstream response to delay GvHD progression. In the context of cell-based therapy, we demonstrated that targeting PKCtheta via a cell-penetrating antibody in induced regulatory T cells (iTregs) generated a novel, highly stable, super-suppressive iTregs by reprogramming their post-transcriptional organization and epigenetic signatures. These reprogrammed iTregs augmented their suppressive activity both in vitro and in vivo, hence, providing a promising cell-based therapy in preventing GvHD. In addition to super-suppressive iTregs, we utilized induced pluripotent stem cell (iPSC)-derived mesenchymal stem cells (MSCs) to prevent GvHD via manipulating PKCtheta signaling in alloreactive T cells. iPSC-MSCs also attenuated GvHD severity and prolonged survival in the humanized model. Altogether, our findings demonstrate the importance of manipulating PKCtheta function in preventing or treating GvHD in the context of BMT therapy for immunological diseases.