Auxin plays critical roles in many developmental processes of plants. The auxin signaling pathway is a series of plant responses to auxin stimuli. However, the functions of many genes in this pathway are still obscure. As auxin receptors, TIR/AFB family genes encode F-Box proteins that directly bind auxin and then transduce the stimulus through the signaling pathway. In this paper, we generated an overexpression line of Auxin-signaling F-Box 6 (OsAFB6) in rice, which largely delayed heading, greatly increased spikelets per panicle and primary branch number and ultimately enhanced grain yield by 50%. OsAFB6 is preferentially expressed in young tissues with stronger meristem activities and suppresses flowering by upregulating OsRR1 and downregulating Ehd1 expression levels. Overexpression of OsAFB6 delayed heading, increased cytokinin (CK) by suppressing the expression level of Gn1a and simultaneously decreased the IAA concentration in the young panicle, which promoted inflorescence meristem development and resulted in large panicles with more spikelets per panicle, primary branches and increased grain yield. It would be a beneficial strategy to generate lines with varied expression levels of OsAFB6 to breed high-yielding cultivars for specific regions that can fully utilize the local sunlight and temperature resources.