MicroRNAs (miRNAs) are a class of non-coding RNAs of approximately 21-nt which play important roles in regulating gene expression in plants. Although many miRNA studies have focused on a few model plants, the miRNAs and their target genes remain largely unknown in pepper, one of the most important crops cultivated worldwide. Here we employed high-throughput small RNA sequencing to extensively identify miRNAs in pepper from 10 different libraries, including leaf, stem, root, flower, and six developmental stage fruits. Based on bioinformatics pipeline, we successfully identified 29 and 35 families of conserved and novel miRNAs, respectively. We noticed that some miRNAs, whose targets were validated experimentally in this study, exhibited prominent changes in expression levels during fruit development stages. From the qRT-PCR analysis of the target mRNAs, including the SBP-transcription factor and F-box protein, we found that expression of these two target mRNAs gradually decreased in general during fruit development and was negatively correlated with the expression of their corresponding miRNAs. The validation of miRNA-directed cleavage of these target mRNAs, combined with the results of qRT-PCR analysis, likely suggests that some miRNAs in pepper may play a role in fruit development. Conclusively, our study first utilizes high-throughput sequencing to identify and characterize conserved and novel miRNAs and their targets in pepper, providing a basis for understanding the functional roles of miRNAs in pepper.