Nucleosome is the building unit of eukaryotic chromatin, the location of histone octamer on the DNA sequence is called nucleosome positioning. With the advent of high throughout technologies such as ChIP-chip and ChIP-seq. large-scale nucleosome positioning atlas of multiple model organisms have been measured, which attract many researchers to investigate nucleosome positioning and its functions on transcriptional regulation. In this paper, we first introduce the concept of nucleosome positioning and summarize the regular patterns of nucleosome positioning on genetic region, then review the major advances of functions of nucleosome positioning on transcriptional initiation, elongation, divergence of expression patterns and alternative splicing.