The lateral transmembrane protein-protein interactions (PPI) have been regarded as “undruggable” despite their importance in many essential biological processes. The homo-trimerization of transmembrane domain 5 (TMD-5) of latent membrane protein 1 (LMP-1) is critical for the constitutive oncogenic activation of the Epstein-Barr virus (EBV). Herein we repurpose the antimicrobial agent pentamidine as a regulator of LMP-1 TMD-5 lateral interactions. The results of ToxR assay, tryptophan fluorescence assay, courmarin fluorescence dequenching assay, and Bis-Tris sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) consistently show pentamidine disrupts LMP-1 TMD-5 lateral interactions. Furthermore, pentamidine inhibits LMP-1 signaling, inducing cellular apoptosis and suppressing cell proliferation in the EBV infected B cells. In contrast, EBV negative cells are less susceptible to pentamidine. This study provides a novel non-peptide small molecule agent for regulating LMP-1 TMD-5 lateral interactions.