Abstract Background Effective tiller number (ETN) has a pivotal role in determination of rice (Oryza sativa L.) grain yield. ETN is a complex quantitative trait regulated by both genetic and environmental factors. Despite multiple tillering-related genes have been cloned previously, few of them have been utilized in practical breeding programs. Results In this study, we conducted a genome-wide association study (GWAS) for ETN using a panel of 490 rice accessions derived from the 3 K rice genomes project. Thirty eight ETN-associated QTLs were identified, interestingly, four of which colocalized with the OsAAP1, DWL2, NAL1, and OsWRKY74 gene previously reported to be involved in rice tillering regulation. Haplotype (Hap) analysis revealed that Hap5 of OsAAP1, Hap3 and 6 of DWL2, Hap2 of NAL1, and Hap3 and 4 of OsWRKY74 are favorable alleles for ETN. Pyramiding favorable alleles of all these four genes had more enhancement in ETN than accessions harboring the favorable allele of only one gene. Moreover, we identified 25 novel candidate genes which might also affect ETN, and the positive association between expression levels of the OsPILS6b gene and ETN was validated by RT-qPCR. Furthermore, transcriptome analysis on data released on public database revealed that most ETN-associated genes showed a relatively high expression from 21 days after transplanting (DAT) to 49 DAT and decreased since then. This unique expression pattern of ETN-associated genes may contribute to the transition from vegetative to reproductive growth of tillers. Conclusions Our results revealed that GWAS is a feasible way to mine ETN-associated genes. The candidate genes and favorable alleles identified in this study have the potential application value in rice molecular breeding for high ETN and grain yield.