Small RNA (sRNA) is a class of noncoding RNA that can silence the expression of target genes. In rice, the majority of characterized sRNAs are within the range of 21 to 24 nucleotides (nt) long, whose biogenesis and function are associated with a specific sets of components, such as Dicer-like (OsDCLs) and Argonaute proteins (OsAGOs). Rice sRNAs longer than 24 nt are occasionally reported, with biogenesis and functional mechanism uninvestigated, especially in a context of defense responses against pathogen infection. By using deep sequencing, we identified a group of rice long small interfering RNAs (lsiRNAs) that are within the range of 25 to 40 nt in length. Our results show that some rice lsiRNAs are differentially expressed upon infection of Rhizoctonia solani, the causal agent of the rice sheath blight disease. Bioinformatic analysis and experimental validation indicate that some rice lsiRNAs can target defense-related genes. We further demonstrate that rice lsiRNAs are neither derived from RNA degradation nor originated as secondary small interfering RNAs (siRNAs). Moreover, lsiRNAs require OsDCL4 for biogenesis and OsAGO18 for function. Therefore, our study indicates that rice lsiRNAs are a unique class of endogenous sRNAs produced in rice, which may participate in response against pathogens.