We show that nucleosomes exert a maximal amount of hindrance to the one-dimensional diffusion of transcription factors (TFs) when they are present between TFs and their cognate sites on DNA. The effective one-dimensional diffusion coefficient of TFs (χTF) decreases with a rise in the free-energy barrier (μNU) of the sliding of nucleosomes as χTF∝exp(−μNU). The average time (ηL) required by TFs to slide over L sites on DNA increases with μNU as ηL∝exp(μNU). When TFs move close to nucleosomes, then they exhibit typical subdiffusion. Nucleosomes can enhance the search dynamics of TFs when TFs are present between nucleosomes and TF binding sites. These results suggest that nucleosome-depleted regions around the cognate sites of TFs are mandatory for efficient site-specific binding of TFs. Remarkably, the genome-wide in vivo positioning pattern of TFs shows a maximum at their specific binding sites where the occupancy of nucleosomes shows a minimum. This could be a consequence of an increasing level of breathing dynamics of nucleosome cores and decreasing levels of fluctuations in the DNA binding domains of TFs as they move across TF binding sites. The dynamics of TFs becomes slow as they approach their cognate sites so that TFs form a tight site-specific complex, whereas the dynamics of nucleosomes becomes rapid so that they quickly pass through the cognate sites of TFs. Several in vivo data sets on the genome-wide positioning pattern of nucleosomes and TFs agree well with our arguments. The retarding effects of nucleosomes can be minimized when the degree of condensation of DNA is such that it can permit a jump size associated with the dynamics of TFs beyond ∼160–180 bp. © 2018 Biophysical Society