MitoCarta3.0 is an updated mitochondrial proteome database that includes sub-organelle localization and pathway annotations. It was developed by integrating multiple sequence features and experimental datasets, including protein mass spectrometry of mitochondria from 14 murine tissues. The database includes 1136 human genes, with 78 new genes added and 100 removed compared to MitoCarta2.0. MitoCarta3.0 provides detailed annotations of sub-mitochondrial localization (matrix, inner membrane, intermembrane space, outer membrane) and assignment to 149 hierarchical 'MitoPathways' spanning seven broad functional categories relevant to mitochondria. The database is freely available at http://www.broadinstitute.org/mitocarta and serves as a community resource for mitochondrial biology and medicine. The database was created through a manual review of the MitoCarta2.0 inventory, with proteins flagged for removal or inclusion and annotated to pathways and sub-compartments. The process involved multiple rounds of iteration and cross-checking by annotators. The sub-mitochondrial localizations of all MitoCarta3.0 proteins were determined by combining experimental datasets and intrinsic protein features. The database also includes annotations of mitochondrial pathways, which were compiled from literature and are more specific than existing broad databases such as KEGG, REACTOME, and GO. MitoCarta3.0 provides a highly specific reference of the mitochondrial proteome with interpretable scores and manual review. MitoCarta3.0 includes annotations of sub-mitochondrial localization and pathway membership for all proteins. The database is freely available and provides a valuable research tool for studying mitochondrial proteins and pathways. It serves as a foundation for the use of high-throughput datasets in molecular biology and human genetics to illuminate the function of understudied or undiscovered mitochondrial proteins. The database is also compared to other mitochondrial databases, such as IMPI and COMPARTMENTS, and is noted for its specificity and manual review. MitoCarta3.0 provides a complementary and mitochondrial-centric annotation of biological functions.MitoCarta3.0 is an updated mitochondrial proteome database that includes sub-organelle localization and pathway annotations. It was developed by integrating multiple sequence features and experimental datasets, including protein mass spectrometry of mitochondria from 14 murine tissues. The database includes 1136 human genes, with 78 new genes added and 100 removed compared to MitoCarta2.0. MitoCarta3.0 provides detailed annotations of sub-mitochondrial localization (matrix, inner membrane, intermembrane space, outer membrane) and assignment to 149 hierarchical 'MitoPathways' spanning seven broad functional categories relevant to mitochondria. The database is freely available at http://www.broadinstitute.org/mitocarta and serves as a community resource for mitochondrial biology and medicine. The database was created through a manual review of the MitoCarta2.0 inventory, with proteins flagged for removal or inclusion and annotated to pathways and sub-compartments. The process involved multiple rounds of iteration and cross-checking by annotators. The sub-mitochondrial localizations of all MitoCarta3.0 proteins were determined by combining experimental datasets and intrinsic protein features. The database also includes annotations of mitochondrial pathways, which were compiled from literature and are more specific than existing broad databases such as KEGG, REACTOME, and GO. MitoCarta3.0 provides a highly specific reference of the mitochondrial proteome with interpretable scores and manual review. MitoCarta3.0 includes annotations of sub-mitochondrial localization and pathway membership for all proteins. The database is freely available and provides a valuable research tool for studying mitochondrial proteins and pathways. It serves as a foundation for the use of high-throughput datasets in molecular biology and human genetics to illuminate the function of understudied or undiscovered mitochondrial proteins. The database is also compared to other mitochondrial databases, such as IMPI and COMPARTMENTS, and is noted for its specificity and manual review. MitoCarta3.0 provides a complementary and mitochondrial-centric annotation of biological functions.