What are the advantages and disadvantages of building nanosystems using one or multiple components? More than 55% of all proteins found in living organisms are multimeric and likely exploit molecular assembly to create new functional entities. However, the specific contribution of molecular assembly to the creation of novel functions remains relatively unexplored at the thermodynamic, kinetic and molecular levels. Here, we use theory and a simple experimental model to determine the design rules for engineering efficient self-assembled, self-regulated nanosystems. Using these rules, we have rationally designed and implemented various regulation mechanisms (e.g., cooperative and anticooperative assembly, self-inhibition, molecular timer) into two model trimeric nanosystems including a complex artificial catalyst. These simple strategies based on molecular assembly have been extensively exploited by natural biosystems and are expected to play a crucial role in the development of future self-regulated nanotechnologies.