Stimuli-responsive polypeptide nanoassemblies exhibit great potentials for cancer nanomedicines because of desirable biocompatibility and biodegradability, unique secondary conformations, varying functionalities, and especially the stimuli-enhanced therapeutic efficacy and reduced side effect. This review introduces the design and fabrication of stimuli-responsive polypeptide nanoassemblies that exhibit endogenous stimuli (e.g., pH, reduction, reactive oxygen species, adenosine triphosphate and enzyme, etc.) and exogenous light stimuli (e.g., UV and near-infrared light), which are biologically related or applied in the clinic. We also discuss the applications and prospects of those stimuli-responsive polypeptide nanoassemblies that might overcome the biological barriers of cancer nanomedicines for in vivo administration. Much more effort is needed to accelerate the second-generation stimuli-responsive polypeptide nanomedicines for clinical transition and applications. This article is categorized under: Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease.