Lithium chloride (LiCl) has been reported to possess antiviral activity against several viruses. In the current study, we assessed the antiviral activity effect of LiCl on ALV-J infection in CEF cells by using real-time PCR, Western blot analysis, IFA and p27 ELISA analysis. Our results showed that both viral RNA copy number and protein level decreased significantly in a dose and time dependent manner. Time-course analysis revealed that the antiviral effect was more pronounced when CEFs were treated at the post infection stage rather than at early absorption or pre-absorption stages. Further experiments demonstrated that LiCl did not affect virus attachment or entry, but rather affected early virus replication. We also found that inhibition of viral replication after LiCl treatment was associated with reduced mRNA levels of pro-inflammatory cytokines. These results demonstrate that LiCl effectively blocked ALV-J replication in CEF cells and may be used as an antiviral agent against ALV-J.