Motivation: The availability of powerful analysis tools will further understanding of genome organization and its relationship to phenotype in dynamical settings. Results: The 4D Nucleome Analysis Toolbox (NAT) is a user-friendly and powerful MATLAB toolbox for time series analysis of genome-wide chromosome conformation capture (Hi-C) data and gene expression (RNA-seq). NAT can load and normalize data, define topologically associating domains, analyse translocations, produce visualization, and study time course data. We provide examples that include time series data sets and karyotypically abnormal cell lines demonstrating the flexibility of NAT. Availability and implementation: https://github.com/laseaman/4D_Nucleome_Analysis_Toolbox. Contact: [email protected].