Guanine quadruplexes are exceptionally stable four stranded structures of nucleic acids that play an important role in regulation of oncogene expression, cell cycle and telomere maintenance. The study of these regulatory pathways is an emergent field and currently little is known about how proteins interact with these structures and control their function. Using a mass spectrometry guided approach, we identified many proteins that can interact with guanine quadruplexes or complexes thereof. From this list we further validated DDX21 can bind directly to RNA guanine quadruplexes through interactions with a unique nucleic acid binding domain in its C-terminus, we did this using a comprehensive suite of biochemical and biophysical techniques. As well as probing binding to quadruplexes we developed a nuclease sensitivity assay that shows that DDX21 binding to quadruplexes destabilizes them sufficiently to allow for strand excision by T1 ribonuclease enzymes, an ability that had only previously been observed by two other proteins. Furthermore, we showed that DDX21 can regulate expression of several important genes in a manner dependent upon its quadruplex binding ability. Together, our results have shed light on the mode by which DDX21 can recognize RNA quadruplex structures and laid the groundwork for understanding the complex network of interactions and enzyme activity which controls quadruplex regulatory function in human pathophysiology.