Abstract: Genomic DNA in the eukaryotic cells is hierarchically folded by histones into chromatin to fit inside the nucleus. DNA wraps around the histone octamers to form nucleosomes, which are connected by linker DNA to form a “beads-on-a-string” nucleosomal array—the primary structure of chromatin. The nucleosomal array is further folded into a condensed 30-nm chromatin fiber. In past three decades, the structure of 30-nm chromatin fiber has been remained as one of the fundamental problems in molecular biology. Based on the early studies of electron microscopy, X-ray crystallography and other biophysical methods, two classic structural models have been hypothesized: the solenoid model and the zig-zag model. Here we review the recent research progresses on the structure of 30-nm chromatin fiber and focus on the recent cryo-EM study of 30-nm chromatin structure which is revealed as a left-handed double helix twisted by tetra-nucleosome units. In addition, we discuss the physiological relevance of 30-nm chromatin fiber in vivo and the perspective on its structural dynamics in epigenetic regulations.