MicroRNAs (miRNAs) represent a newly identified class of non-protein-coding approximately 22 nt small RNAs which play important roles in multiple biological processes by degrading targeted mRNAs or repressing mRNA translation. Here we present an expressed sequence tag (EST)-based combined approach for the detection of novel porcine miRNAs. This was initiated by using previously known miRNA sequences from Homo sapiens (human) and Mus musculus (mouse) to blast the databases of Sus scrofa (pig) EST. A total of 65 new miRNAs were detected following a range of filtering criteria. Using these new potential miRNA sequences, we further obtained the publicly available porcine mRNA database from NCBI and detected 48 586 potential target hits using a software RNA hybrid. So far, compared to human and mouse, fewer miRNAs (only 54 miRNAs) were identified in Sus scrofa species. These 65 new miRNAs and their targets in pig have been run through miRHelper to yield data that may help us better understand the possible role of miRNAs in regulating the growth and development of pigs. These findings suggest that EST analysis is a good alternative strategy for identifying new miRNA candidates, their targets and other genes.