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Abstract In Escherichia coli, RNase E is the key enzyme for RNA processing and mRNA degradation. Despite the conserved function across bacteria, the domain composition of RNase E varies significantly among species, possibly affecting the enzyme’s subcellular localization, mobility, and function. In this work, we used super-resolution microscopy to find that 93% of RNase E is localized to the membrane in E. coli and exhibits slow diffusion comparable to polysomes diffusing in the cytoplasm. By replacing the native amphipathic membrane targeting sequence (MTS) with a transmembrane motif, we discovered that the MTS results in slower diffusion and stronger membrane binding than a transmembrane motif. Additionally, the evolutionarily divergent C-terminal domain (CTD) was shown to grant slow diffusion of RNase E but to weaken its membrane binding. By analyzing how membrane localization and diffusion of RNase E affect mRNA degradation rates in vivo, we provide new insights into RNase E’s role in the spatiotemporal organization of RNA processes in bacterial cells.