Abstract Summary The PICKLE 3.0 upgrade refers to the enrichment of this human protein–protein interaction (PPI) meta-database with the mouse protein interactome. Experimental PPI data between mouse genetic entities are rather limited; however, they are substantially complemented by PPIs between mouse and human genetic entities. The relational scheme of PICKLE 3.0 has been amended to exploit the Mouse Genome Informatics mouse–human ortholog gene pair collection, enabling (i) the extension through orthology of the mouse interactome with potentially valid PPIs between mouse entities based on the experimental PPIs between mouse and human entities and (ii) the comparison between mouse and human PPI networks. Interestingly, 43.5% of the experimental mouse PPIs lacks a corresponding by orthology PPI in human, an inconsistency in need of further investigation. Overall, as primary mouse PPI datasets show a considerably limited overlap, PICKLE 3.0 provides a unique comprehensive representation of the mouse protein interactome. Availability and implementation PICKLE can be queried and downloaded at http://www.pickle.gr. Supplementary information Supplementary data are available at Bioinformatics online.