MicroRNAs (miRNAs) are about 22 nucleotides regulatory non-coding RNAs that play versatileroles in reprogramming plant responses to biotic and abiotic stresses. However, it remains unknown whether miRNAs confer the resistance to necrotrophic fungus Rhizoctonia solani in rice. To investigate whether miRNAs regulate the resistance to R. solani, we constructed 12 small RNA libraries from susceptible and resistant rice cultivars treated with water/pathogen at 5 h post inoculation (hpi), 10 hpi and 20 hpi, respectively. By taking the advantage of next-generation sequencing, we totally collected 400–450 known miRNAs and 450–620 novel miRNAs from the libraries. Expression analysis of miRNAs demonstrated different patts for known and novel miRNAs upon R. solani challenge. Thirty-four miRNA families were identified to be expressed specifically in rice, and most of them were involved in plant disease resistance. A particular Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis result revealed that a great majority of target genes of regulated miRNAs belonged to the pathway of plant-pathogen interaction. Moreover, miR444b.2, miR531a, mir1861i, novel_miR1956 and novel_miR135 conferred response to R. solani infection confirmed by North blot. Our global understanding of miRNA profiling revealed that the regulation of miRNAs may be implicated in the control of rice immunity to R. solani. Analysis of the expression of miRNAs will offer the community with a direction to generate appropriate strategies for controlling rice sheath blight disease.