Collectively, we demonstrated that methadone inhibited the expression of IFN-β and IFN-λ2 in primary macrophages with/without HIV infection, which further regulated the subsequent restriction factors (APOBEC3F, APOBEC3G, and MxB) and other anti-HIV miRNAs (miR-28, miR-125b, miR-150, and miR-155). Here, APOBEC3G is linked to HIV infectious disease.