Promoter-proximal pausing of RNA polymerase Ⅱ (RNAPⅡ) is a critical step for the precise regulation of gene expression. Here, we provide evidence of promoter-proximal pausing-like distributions of RNAPⅡ in S. cerevisiae using PRO-seq experiments. Furthermore, we find that knockdown of the chromatin remodeler Ino80p causes the transition of RNAPⅡ pausing from the main pausing site to the alternative pausing site. In addition, these Ino80p-dependent genes exhibit the +1 nucleosome closely associated with the alternative pausing site, which implied the suppression of the alternative pausing site in a nucleosome context-dependent manner. Ino80p knockdown results in the increase of nucleosome fuzziness and the decrease of nucleosome stability for these genes, suggesting the role of the Ino80 complex to regulate the +1 nucleosome is highly correlated with the proper localization of RNAPⅡ pausing. Moreover, we observed similar INO80-dependent RNAPⅡ pausing site determination in mouse embryonic stem cells (mESCs). Based on our collective results, we hypothesize that the chromatin remodeler Ino80p plays a highly conserved role in regulating early RNAPⅡ elongation to establish intact pausing in various organisms, from budding yeast to mouse.