Plant microRNAs (miRNAs) have been commonly investigated during many years. Hundreds of miRNAs have been identified in many different plant species but there is very little information about the function of sesame (Sesamum indicum L.) miRNAs. For this purpose, in silico prediction of novel sesame miRNAs based on BLAST searches of the expressed sequence tag database was performed, using stringent criterias for miRNA annotation. The secondary structures of their precursor sequences, potential target genes of conserved and novel miRNAs were predicted and subjected to Gene Ontology (GO) annotation. mir447 and mir8140 were reported for the first time in sesame. Enrichment analysis of the GO with biological processes, cellular component and molecular functions revealed that these target genes were potentially involved in different metabolic pathways such as transcription factors, metabolism, growth and development, stress-related and even plant hormones. Results are valuable for figure out the gene regulation mechanism in sesame, using in the medicinal aspect of this plant species. Furthermore, these miRNAs and their profiled targets could provide the improvement of regulation and management, and even development of desirable traits in this plant.