Porcine reproductive and respiratory syndrome virus (PRRSV) is an important pathogen that causes economically huge losses to the pig industry worldwide. Current control of PRRSV infection remains inadequate although various means have been implemented. Thus, investigating novel antiviral therapeutics to combat PRRSV infection is essential. In the present study, the antiviral effect in vitro of 25-hydroxycholesterol (25HC) against PRRSV was investigated. Cell viability assay was performed to examine the impact of 25HC on the cell viability. Indirect immunofluorescence assay and virus titration were utilized to evaluate the levels of PRRSV growth. Viral attachment assay, penetration assay and release assay were conducted to investigate the antiviral mechanism of 25HC against PRRSV. Real-time RT-PCR assay was used to analyse the effect of 25HC on the genome synthesis of PRRSV. We demonstrated that the growth of PRRSV was significantly inhibited in 25HC-pretreated cells and PRRSV-infected cells by 25HC. Moreover, 25HC could impair the attachment and entry of PRRSV in vitro, but not affect viral genome synthesis and virion release. Our findings clearly indicate that 25HC can exert antiviral effect against PRRSV infection in vitro, suggesting that 25HC might be a novel potential agent to control PRRSV infection.