The complex gene regulatory network underlying maize tiller development remains largely unknown. Here we identified two major quantitative trait loci (QTLs) for tiller number, Tin8 on chromosome 8 and the previously known Tb1 on chromosome 1, in a population derived from a teosinte-maize cross. Map-based cloning and association mapping revealed that Tin8 corresponding to Zcn8 encoding a PEBP-related kinase, is down-regulated in transcription and thus results in decreased tiller number. Strong interaction between Tin8 and the key gen Tb1 was detected for tiller number. Further RNA-seq analysis showed that the expressions of 13 genes related to tiller development were controlled by Tin8. Our results support the existence of a complex gene regulatory network for the outgrowth of maize tiller bud, in which Zcn8 controls 13 tiller-related genes including four genes for hormonal responses. Especially, Zcn8 represses Gt1, D14 and Tru1, through the interaction of Tb1.