MitoCarta3.0 is an updated catalog of the mammalian mitochondrial proteome, now including sub-organelle localization and pathway annotations. The catalog was developed by manually reviewing and updating the MitoCarta2.0 inventory, which was initially compiled using Bayesian integration of multiple sequence features and experimental datasets. MitoCarta3.0 includes 1136 human genes, with 78 new additions and 100 removals based on literature review and experimental evidence. The catalog provides annotations for sub-mitochondrial localization (matrix, inner membrane, intermembrane space, outer membrane) and assignment to 149 hierarchical 'MitoPathways' spanning seven broad functional categories relevant to mitochondria. These annotations are freely available at http://www.broadinstitute.org/mitocarta and serve as a valuable resource for mitochondrial biology and medicine. The accuracy of sub-mitochondrial localizations was assessed by comparing annotations to a recently published mitochondrial proximity interaction network, finding 98% consistency. MitoCarta3.0 also includes mitochondria-centric pathway annotations, which are more specific and accurate than those provided by broader databases like KEGG and GO. Despite its limitations, MitoCarta3.0 is a valuable tool for studying mitochondrial proteins and pathways.MitoCarta3.0 is an updated catalog of the mammalian mitochondrial proteome, now including sub-organelle localization and pathway annotations. The catalog was developed by manually reviewing and updating the MitoCarta2.0 inventory, which was initially compiled using Bayesian integration of multiple sequence features and experimental datasets. MitoCarta3.0 includes 1136 human genes, with 78 new additions and 100 removals based on literature review and experimental evidence. The catalog provides annotations for sub-mitochondrial localization (matrix, inner membrane, intermembrane space, outer membrane) and assignment to 149 hierarchical 'MitoPathways' spanning seven broad functional categories relevant to mitochondria. These annotations are freely available at http://www.broadinstitute.org/mitocarta and serve as a valuable resource for mitochondrial biology and medicine. The accuracy of sub-mitochondrial localizations was assessed by comparing annotations to a recently published mitochondrial proximity interaction network, finding 98% consistency. MitoCarta3.0 also includes mitochondria-centric pathway annotations, which are more specific and accurate than those provided by broader databases like KEGG and GO. Despite its limitations, MitoCarta3.0 is a valuable tool for studying mitochondrial proteins and pathways.