We explored strategies to modulate epithelial tight junctions for barrier protection and for drug delivery. Our findings indicate that toll-like receptor 2 (TLR2) stimulation in human bronchial epithelial cells increased tight junction barrier function by increasing the expression of claudin-1, and through modulation of PKCζ activity. We designed and characterized cell permeable short peptides that can transiently open tight junctions. The peptides induced redistribution of tight junction proteins claudin-1 and ZO-1 into the intracellular compartment thereby inducing a transient increase in paracellular permeability. We investigated the lead short peptide (SG-01) for its ability to act as an anti-tumor adjuvant and mucosal vaccine adjuvant. The peptide and its combination with a cytotoxicity drug gefitinib showed a significant improvement in the reduction of tumor size in a 3D tumor model. A preclinical study in mice showed that SG-01 significantly improved immunogenicity of ovalbumin probably by improving its permeation across nasal mucosa.