Given the growing evidence for a role of interleukin-32 (IL-32) in the immune response to HIV-1 infection and its interplay with type I and III interferons (IFNs), we studied the gene expression of IL-32 isoforms (α and nonα) in untreated chronically HIV-1-infected patients and in gender- and age-matched healthy individuals. To further characterize both the anti-HIV properties of IL-32 and the cytokine's relationship with host antiviral innate immune responses, we evaluated whether IL-32 can induce ex vivo the expression of antiviral IFN-induced genes (ISGs), namely myxovirus resistance A (MxA), and apolipoprotein B mRNA-editing enzyme catalytic (APOBEC)3G and APOBEC3F. We also investigated whether in vivo IL-32 (α and nonα) mRNA levels were correlated with those of MxA and APOBEC3G/3F. Results indicated that IL-32 (α and nonα) mRNA levels were significantly higher in HIV-1-infected patients than in healthy individuals. Furthermore, IL-32 (α and nonα) mRNA levels correlated negatively with HIV RNA levels, but not with the CD4(+) T-cell count. Our ex vivo studies disclosed that ISGs mRNA levels were increased after IL-32γ treatment of peripheral blood mononuclear cells. Interestingly, significant positive correlations were found between transcript levels of both IL-32α and IL-32nonα and those of MxA and APOBEC3G/3F in untreated chronically HIV-1-infected patients. Overall, our results demonstrated that IL-32 isoforms are highly expressed during chronic HIV-1 infection and that IL-32 could have a central role in the antiviral immune response against HIV-1.