Betalains are water-soluble nitrogen-containing pigments with multiple bioactivities. Pitaya is the only large-scale commercially grown fruit containing abundant betalains for consumers. However, the upstream regulators in betalain biosynthesis are still not clear. In this study, HmoWRKY40, a novel WRKY transcription factor, was obtained from the transcriptome data of pitaya (). HmoWRKY40 is a member of the Group IIa WRKY family, containing a conserved WRKY motif, and it is located in the nucleus. The betalain contents and expression levels of HmoWRKY40 increased rapidly during the coloration of pitaya and reached their maximums on the 23rd day after artificial pollination (DAAP). Yeast one-hybrid and transient expression assays showed that HmoWRKY40 could bind and activate the promoter of . Silencing the gene resulted in a significant reduction of betacyanin contents. These results indicate that HmoWRKY40 transcriptionally activates which is involved in the regulation of pitaya betalain biosynthesis. The results of the present study provide new regulatory networks related to betalain biosynthesis in pitaya.