AbstractIntracellular bacteria in latent or dormant states tolerate high‐dose antibiotics. Fighting against these opportunistic bacteria has been a long‐standing challenge. Herein, the design of a cascade‐targeting drug delivery system (DDS) that can sequentially target macrophages and intracellular bacteria, exhibiting on‐site drug delivery, is reported. The DDS is fabricated by encapsulating rifampicin (Rif) into mannose‐decorated poly(α‐N‐acryloyl‐phenylalanine)‐block‐poly(β‐N‐acryloyl‐d‐aminoalanine) nanoparticles, denoted as Rif@FAM NPs. The mannose units on Rif@FAM NPs guide the initial macrophage‐specific uptake and intracellular accumulation. After the uptake, the detachment of mannose in acidic phagolysosome via Schiff base cleavage exposes the d‐aminoalanine moieties, which subsequently steer the NPs to escape from lysosomes and target intracellular bacteria through peptidoglycan‐specific binding, as evidenced by the in situ/ex situ co‐localization using confocal, flow cytometry, and transmission electron microscopy. Through the on‐site Rif delivery, Rif@FAM NPs show superior in vitro and in vivo elimination efficiency than the control groups of free Rif or the DDSs lacking the macrophages‐ or bacteria‐targeting moieties. Furthermore, Rif@FAM NPs remodel the innate immune response of the infected macrophages by upregulating M1/M2 polarization, resulting in a reinforced antibacterial capacity. Therefore, this biocompatible DDS enabling macrophages and bacteria targeting in a cascade manner provides a new outlook for the therapy of intracellular pathogen infection.