Nannocystins are a family of 21-membered cyclodepsipeptides with excellent anticancer activity. However, their macrocyclic architecture poses a significant challenge to structure modification. Herein, this issue is addressed by leveraging the strategy of post-macrocyclization diversification. In particular, a novel serine-incorporating nannocystin was designed so that its appending hydroxyl group could diversify into a wide variety of side chain analogues. Such effort facilitated not only structure-activity correlation at the subdomain of interest, but also the development of a macrocyclic coumarin-labeled fluorescence probe. Uptake experiments indicated good cell permeability of the probe, and endoplasmic reticulum was identified as its subcellular localization site.