Immune tolerance induced by avian leukosis virus subgroup J (ALV-J) is a prerequisite for tumorigenesis. Although we had reported that B cell anergy induced by ALV-J was the main reason for immune tolerance, the molecular mechanism still remains unclear. Here, we found SU protein of ALV-J interacted with tyrosine kinase Lyn (a key protein in BCR signaling pathway) by confocal laser scanning microscopy and co-immunoprecipitation test, which suggested that Lyn might play an important role in B cell anergy induced by ALV-J. Correspondingly, the mRNA and protein level of Lyn was significantly up-regulated in B cells after ALV-J infection. Subsequently, the phosphorylated protein levels of Lyn at Tyr507 site were significantly up-regulated in ALV-J-infected B cells after BCR signal activation, but the phosphorylated protein level of Syk (a direct substrate of Lyn) at Tyr525/526 site, Ca2+ flux, and NF-κB p65 protein level were significantly down-regulated. Interestingly, the phosphorylated protein level of Syk at Tyr525/526 site, Ca2+ flux, and NF-κB p65 protein level were both significantly retrieved after the shLyn treatment in B cells infected by ALV-J. In summary, these results indicated that ALV-J activated the negative regulatory effect of phosphorylated Lyn protein at 507 site in BCR signal transduction pathway and then mediated B cell anergy, which will provide a new insight for revealing the pathogenesis of immune tolerance induced by ALV-J.