MicroRNAs (miRNAs) are small non-coding regulatory RNAs that play key roles in many diverse biological processes such as spermatogenesis. However, no study has been performed on the miRNA transcriptome of developing porcine testes. Here, we employed Solexa deep sequencing technology to extend the repertoire of porcine testis miRNAs and extensively compare the expression patterns of the sexually immature and mature porcine testes. Solexa sequencing of two small RNA libraries derived from immature (30 days) and mature (180 days) pig testis samples yielded over 25 million high-quality reads. Overall, the two developmental stages had significantly different small RNA compositions. A custom data analysis pipeline identified 398 known and/or homologous conserved porcine miRNAs, 15 novel pig-specific miRNAs, and 56 novel candidate miRNAs. We further observed multiple mature miRNA variants (isomiRs) and identified a new bidirectional transcribed miRNA locus, ssc-mir-181a. One hundred twenty-two miRNAs were differentially expressed in the immature and mature testes, and 10 were validated using quantitative RT-PCR. Furthermore, GO and KEGG pathway analyses of the predicted miRNA targets further illustrate the likely roles for these differentially expressed miRNAs in spermatogenesis. This study is the first comparative profile of the miRNA transcriptome in immature and mature porcine testes using a deep sequencing approach, and it provides a useful resource for future studies on the role of miRNAs in spermatogenesis and male infertility treatment. microRNA profiling and discovery in two small RNA cDNA libraries derived from sexually immature (30-day) and mature (180-day) pig testes.