In the emerging field of photopharmacology, synthetic photoswitches based on reversible photochemical reactions are fused to bioactive molecules. Azobenzene derivatives, which can undergo trans-cis photoisomerization, are typical photoswitches. Most azobenzene-based photochemical tools are active in the thermodynamically stable trans, but not cis, form. cis-Active photochemical tools would be ideal because they can be "initially inactive and active after light illumination" in a reversible mode only by light illumination. However, only a few rational strategies for constructing such "lit-active" photopharmacological tools has been developed. Herein, we report a rationally designed lit-active photoswitchable inhibitor targeting centromere-associated protein E (CENP-E). Using the lit-active inhibitor, we were able to photoregulate CENP-E-dependent mitotic chromosome location in cells. This study provides a framework to facilitate further progress in the development of photopharmacological tools.