Several factors are known to determine chromatin organization. However, the role of non-canonical DNA structure has not been studied in this context. Our recent observations indicated a widespread role of a particular non-canonical DNA structure, the G-quadruplex, or G4 motifs, in gene regulation. Herein, we first analyzed potential G4 (PG4) motif occurrence vis-à-vis nucleosome occupancy signals. Genome-wide analysis using reported nucleosome positions in Saccharomyces cerevisiae and human (ENCODE regions and 3239 promoters) showed exclusion of nucleosomes by regions that have a relatively high density of PG4 motifs (P < 0.0001). This was supported by the enrichment of PG4 motifs within DNase I hypersensitive sites, which generally exclude nucleosomes. Based on these, we asked whether PG4 motifs had any distinct regulatory function. Two non-overlapping gene-sets in humans were tested-with PG4-enriched (Set I) or nucleosome-enriched (Set II) promoters. Gene-sets I and II were enriched in distinct functions: apoptosis and cellular signaling vs. development and immunity-related, respectively (P < 0.01). Sets I and II also showed different tissue-specific expression in 35 normal human tissues. In S. cerevisiae, we noted significant enrichment of PG4 motif regulated genes in cellular response to heat-shock, while genes with nucleosome-enriched promoters were not significantly represented. Our results show a structural motif as a possible nucleosome exclusion signal for the first time, and predict an alternate/additional regulatory role of G4 motifs, which could be distinct from gene regulation by remodeling of nucleosomes.