Previously, we reported that little canonical (H3.1-H4)(2) tetramers split to form "hybrid" tetramers consisted of old and new H3.1-H(4) dimers, but approximately 10% of (H3.3-H4)2 tetramers split during each cell cycle. In this report, we mapped the H3.3 nucleosome occupancy, the H3.3 nucleosome turnover rate and H3.3 nucleosome splitting events at the genome-wide level. Interestingly, H3.3 nucleosome turnover rate at the transcription starting sites (TSS) of genes with different expression levels display a bimodal distribution rather than a linear correlation towards the transcriptional activity, suggesting genes are either active with high H3.3 nucleosome turnover or inactive with low H3.3 nucleosome turnover. H3.3 nucleosome splitting events are enriched at active genes, which are in fact better markers for active transcription than H3.3 nucleosome occupancy itself. Although both H3.3 nucleosome turnover and splitting events are enriched at active genes, these events only display a moderate positive correlation, suggesting H3.3 nucleosome splitting events are not the mere consequence of H3.3 nucleosome turnover. Surprisingly, H3.3 nucleosomes with high splitting index are remarkably enriched at enhancers in a cell-type specific manner. We propose that the H3.3 nucleosomes at enhancers may be split by an active mechanism to regulate cell-type specific transcription.