CoryneRegNet is the reference database and analysis platform for corynebacterial gene regulatory networks. It provides web-based access to integrated data on gene regulatory interactions of corynebacteria relevant to human medicine and biotechnology, Escherichia coli and Mycobacterium tuberculosis. To facilitate the analysis and reconstruction of the corresponding networks, CoryneRegNet provides user-friendly interfaces for bioinformatics analysis and network visualization tools. This protocol describes four major workflows: (1) querying the regulatory network of a gene of interest, (2) prediction and interspecies transfer of gene regulatory interactions, (3) visualization and comparison of predicted or known networks and (4) integration of gene expression data analysis and visualization. This protocol guides the user through the most important features of CoryneRegNet and takes 45-60 min to complete.