Stimuli-responsive hydrogels are intriguing biomaterials useful for spatiotemporal controlled release of drugs, cells, and biological cues, cell engineering for various applications, and medical diagnosis. To date, many physical and chemical stimuli-responsive polymer hydrogels have been developed by chemical modification of polymer chains and cross-linking points. In particular, conjugation with biomolecules to polymers produced promising biomolecule-responsive hydrogels. These examples clearly indicate high potentials of stimuli-responsive hydrogels as promising biomaterials. In addition to polymer hydrogels, supramolecular hydrogels formed by the assembly of small molecules (hydrogelators) via noncovalent interactions have also been regarded as unique and promising soft materials due to their flexible programmability in rendering them stimuli-responsive with the larger macroscopic change (i.e., gel-sol transition). This Account describes our strategies for the rational design of stimuli-responsive supramolecular hydrogels and their biological applications. Following the detailed structural analysis of a lead hydrogelator that clearly indicates the appropriate sites for incorporation of stimuli-responsive modules, we designed supramolecular hydrogels capable of responding to simple physical (thermal and light) and chemical (pH and metal ions) stimuli. More importantly, biomolecule-responsive hydrogels were successfully developed by supramolecularly mimicking the complex yet well-ordered structures and functions of live cells containing multiple components (a cell-mimicking approach). Development of biomolecule-responsive supramolecular hydrogels has been difficult as the conventional strategy relies on the chemical incorporation of stimuli-responsive modules, owing to the lack of modules that can effectively respond to structurally diverse and complicated biomolecules. Inspired by natural systems where functional compartments (e.g., cell organelles) sophisticatedly interact with each other, we sought to integrate the two distinct microenvironments of supramolecular hydrogels (the aqueous cavity surrounded by fibers and the fluidic hydrophobic fiber domain) with other functional materials (e.g., enzymes, peptides or proteins, fluorescent chemosensors, or inorganic porous or layered nanomaterials) for biomolecule responses. In situ fluorescence microscopy imaging clearly demonstrated that chemical isolation and crosstalk are highly successful between the integrated microenvironments in supramolecular hydrogels, similar to organelles in living cells, which allow for the construction of unique optical response and sensing systems for biomolecules. Furthermore, programmed hybridization of our chemically reactive hydrogels with appropriate enzymes can provide an unprecedented universal platform for biomolecule-degradable supramolecular hydrogels. Such biomolecule-responsive hydrogels are a potentially promising tool for user-friendly early diagnostics and on-demand drug-releasing soft materials. We expect that our rational design strategies for stimuli-responsive supramolecular hydrogels by modification of chemical structures and hybridization with functional materials will inspire scientists in various fields and lead to development of novel soft materials for biological applications.