This manuscript reports the fine mapping of a novel QTL, qAC2 controlling the low amylose in rice. The action mechanism of the qAC2 is also investigated by the analysis of genetic interactions to Wx (a), Wx (b), du1, du2 and du3. Amylose content of the rice (Oryza sativa L.) endosperm greatly affects starch properties and eating quality of cooked rice. Seeds of japonica rice cultivar Kuiku162 have low amylose content (AC) and good eating quality. Our analysis revealed a novel QTL, designated as qAC2 that contributed to the low AC of Kuiku162. qAC2 was fine mapped within a 74.9-kb region between two insertion and deletion markers, KID3001 and KID5101, on the long arm of chromosome 2. Seven genes are predicted in this region, but none of them is known to be related to the regulation of AC. The AC of a near-isogenic line (NIL110) carrying qAC2 (Kuiku), the Kuiku162 allele of qAC2, in the genetic background of japonica cultivar Itadaki was lower by 1.1% points than that of Itadaki. The chain length distributions of amylopectin were similar in NIL110 and Itadaki; therefore, the low AC of NIL110 was caused by a decrease in the actual AC, but not by a difference in the amylopectin structure. The interaction analyses revealed that qAC2 (Kuiku) has epistatic interaction with Wx (a). The qAC2 (Kuiku) has epistatic interactions with two loci, du1 and du2, on Wx (b), whereas the genetic effect of qAC2 (Kuiku) has additive to that of du3 on Wx (b). Thus, similar to du1 and du2, qAC2 may have a function related to Wx (b) mRNA splicing.