During the last century, 2D cell cultures have been the tool most widely used to study cancer biology, drug discovery, genomics, and the regulation of gene expression at genetic/epigenetic levels. However, this experimental approach has limitations in faithfully recreating the microenvironment and cellular processes occurring in tumors. For these reasons, 3D cell cultures have recently been implemented to optimize the conditions that better recreate the biological and molecular features of tumors, including cell–cell and cell–extracellular matrix (ECM) interactions, growth kinetics, metabolic activities, and the development of gradients in the cellular microenvironment affecting the availability of oxygen and nutrients. In this sense, tumor cells receive stimuli from the local environment, resulting in significant changes in their signaling pathways, gene expression, and transcriptional and epigenetic patterns. In this review, we discuss how different types of 3D cell culture models can be applied to characterize the epigenetic footprints of cancer cell lines, emphasizing that DNA methylation patterns play an essential role in the emergence and development of cancer. However, how 3D cancer cell cultures remodel the epigenetic programs is poorly understood, with very few studies in this emerging topic. Here, we have summarized the studies on the reprogramming of the epigenetic landscape of DNA methylation during tumorigenesis and discuss how it may be affected by microenvironmental factors, specifically in 3D cell cultures.