Trilobolide (Tb) is a potent natural counterpart of thapsigargin, which has shown promising results in cancer clinical trials. Here, we report a rational approach to study intracellular localization and biological activity of this sesquiterpene lactone. We conjugated Tb with a green-emitting Bodipy dye attached by alternative linkers of different lengths. The live-cell imaging of the prepared bioconjugates brought clear evidence that Tb-Bodipy localized in the endoplasmic reticulum (ER) of various cancer cell lines. The localization signal was compared with ER-specific dyes. Cytotoxicity of Tb conjugates and impact on the mitochondrial physiology and nitric oxide release were also studied. The nitric oxide production and cytokine secretion in rat peritoneal cells indicate immunobiological potential of these lactone bioconjugates. In summary, our Tb-Bodipy conjugates could help us to reveal the molecular mechanism of trilobolide for its further potential use in biomedical applications.