Large proportion of human genome produces non-coding RNA (ncRNA), including divergent antisense transcript at the promoters. However, the regulatory mechanism of divergent antisense transcription has not been elucidated. In this study, we identified novel complex composed of ZC3H4, WDR82 and CK2 (ZWC complex) that plays an important role in divergent antisense transcription regulation. ZC3H4-WDR82 recognizes S5p RNAPII C-terminal domain (CTD), and all the ZWC complex subunits are co-localized in transcription start sites (TSSs) of active genes. Depletion of ZC3H4 induces increase of divergent antisense transcription in a subset of genes. Furthermore, we identified that the ZWC complex phosphorylates the N-terminal region of SPT5, a subunit of elongation factor DSIF, and showed that this SPT5-phosphorylation is crucial for the regulation of divergent antisense transcription. Taken together, our study reported the novel relationship between the ZWC complex and DSIF, and provided a new insight into the mechanism by which the direction of transcription is determined in the early stage of transcription.