Entangled proteins have attracted significant research interest. Herein, we report the first rationally designed lasso proteins, or protein 1rotaxanes, by using a p53dim‐entwined dimer for intramolecular entanglement and a SpyTag‐SpyCatcher reaction for side‐chain ring closure. The lasso structures were confirmed by proteolytic digestion, mutation, NMR spectrometry, and controlled ligation. Their dynamic properties were probed by experiments such as end‐capping, proteolytic digestion, and heating/cooling. As a versatile topological intermediate, a lasso protein could be converted to a rotaxane, a heterocatenane, and a “slide‐ring” network. Being entirely genetically encoded, this robust and modular lasso‐protein motif is a valuable addition to the topological protein repertoire and a promising candidate for protein‐based biomaterials. Artificially designed lasso proteins were modularly synthesized in cellulo based on assembly–reaction synergy. This enables the synthesis of protein (pseudo)rotaxanes, protein heterocatenanes, and protein‐based “slide‐ring” hydrogels via topological transformation.