Protein subcellular localization (SCL) is important for understanding protein function, genome annotation, and has practical applications such as identification of potential vaccine components or diagnostic/drug targets. PSORTdb (http://db.psort.org) comprises manually curated SCLs for proteins which have been experimentally verified (ePSORTdb), as well as pre-computed SCL predictions for deduced proteomes from bacterial and archaeal complete genomes available from NCBI (cPSORTdb). We now report PSORTdb 3.0. It features improvements increasing user-friendliness, and further expands both ePSORTdb and cPSORTdb with a focus on improving protein SCL data in cases where it is most difficult-proteins associated with non-classical Gram-positive/Gram-negative/Gram-variable cell envelopes. ePSORTdb data curation was expanded, including adding in additional cell envelope localizations, and incorporating markers for cPSORTdb to automatically computationally identify if new genomes to be analysed fall into certain atypical cell envelope categories (i.e. Deinococcus-Thermus, Thermotogae, Corynebacteriales/Corynebacterineae, including Mycobacteria). The number of predicted proteins in cPSORTdb has increased from 3,700,000 when PSORTdb 2.0 was released to over 13,000,000 currently. PSORTdb 3.0 will be of wider use to researchers studying a greater diversity of monoderm or diderm microbes, including medically, agriculturally and industrially important species that have non-classical outer membranes or other cell envelope features.