Abstract: The objective of this study was to analyze the secondary structure and function of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) 5′ UTR. Based on the PRRSV type 2 full-length infectious cDNA clone pAPRRS, a series of mutants which can modify a conserved stem structure were obtained through PCR-based mutagenesis. The mutants are transfected into MARC-145 cells for cytopathic effect (CPE) observation. Meanwhile, the indirect immunofluorescence analysis (IFA) and Northern blot analysis show the genomic replication and subgenomic transcription for the rescued viruses. In addition, the growth features are also detected by plaque morphology compared with the parental virus. It was found that the secondary structure of the conserved stem loop in PRRSV 5′ UTR wasn’t essential for virus rescue. Novertheless, the primary sequence of the stem structure is functional for viral replication. In addition, it is demonstrated that a key nucleotide in PRRSV 5′ UTR directly regulate the viral replication. All results demonstrate that the essential sequences in PRRSV 5′ UTR are functional in viral genomic eplication.