Several members of the APOBEC3 DNA cytosine deaminase family can potently inhibit Vif-deficient human immunodeficiency virus type 1 (HIV-1) by catalyzing cytosine deamination in viral cDNA and impeding reverse transcription. HIV-1 counteracts restriction with the virally encoded Vif protein, which targets relevant APOBEC3 proteins for proteasomal degradation. HIV-1 Vif is optimized for degrading the restrictive human APOBEC3 repertoire, and, in general, lentiviral Vif proteins specifically target the restricting APOBEC3 enzymes of each host species. However, simian immunodeficiency virus SIVmac239 Vif elicits a curiously wide range of APOBEC3 degradation capabilities that include degradation of several human APOBEC3s and even human APOBEC3B, a non-HIV-1-restricting APOBEC3 enzyme. To better understand the molecular determinants of the interaction between SIVmac239 Vif and human APOBEC3B, we analyzed an extensive series of mutants. We found that SIVmac239 Vif interacts with the N-terminal domain of human APOBEC3B and, interestingly, that this occurs within a structural region homologous to the HIV-1 Vif interaction surface of human APOBEC3G. An alanine scan of SIVmac239 Vif revealed several residues required for human APOBEC3B degradation activity. These residues overlap HIV-1 Vif surface residues that interact with human APOBEC3G and are distinct from those that engage APOBEC3F or APOBEC3H. Overall, these studies indicate that the molecular determinants of the functional interaction between human APOBEC3B and SIVmac239 Vif resemble those between human APOBEC3G and HIV-1 Vif. These studies contribute to the growing knowledge of the APOBEC-Vif interaction and may help guide future efforts to disrupt this interaction as an antiviral therapy or exploit the interaction as a novel strategy to inhibit APOBEC3B-dependent tumor evolution.IMPORTANCE Primate APOBEC3 proteins provide innate immunity against retroviruses such as HIV and SIV. HIV-1, the primary cause of AIDS, utilizes its Vif protein to specifically counteract restrictive human APOBEC3 enzymes. SIVmac239 Vif exhibits a much wider range of anti-APOBEC3 activities that includes several rhesus macaque enzymes and extends to multiple proteins in the human APOBEC3 repertoire, including APOBEC3B. Understanding the molecular determinants of the interaction between SIVmac239 Vif and human APOBEC3B adds to existing knowledge on the APOBEC3-Vif interaction and has potential to shed light on what processes may have shaped Vif functionality over evolutionary time. An intimate understanding of this interaction may also lead to a novel cancer therapy because, for instance, creating a derivative of SIVmac239 Vif that specifically targets human APOBEC3B could be used to suppress tumor genomic DNA mutagenesis by this enzyme, slow ongoing tumor evolution, and help prevent poor clinical outcomes.