Background: Antiretroviral therapy (ART) significantly improves the life span of people living with HIV/AIDS (PLWHA). However, HIV- associated cardiovascular diseases (CVDs) remain one of the leading causes of heart failure and comorbidity in PLWHA. Modern ART regimens reduce viral loads to undetectable levels in HIV+ patients while being less toxic. Nevertheless, HIV proteins are present in the plasma of PLWHA. The HIV protein, Nef, has been found circulating in the plasma of ART-treated patients within extracellular vesicles and in association with immune cells. We hypothesized that circulating HIV Nef proteins may cause an adverse effect on various organ functions. To further understand the effect of Nef on differing organ functions, we have generated a transgenic mouse model which expresses the Nef protein in the presence of Cre recombinase. Methods and Results: Nef transgenic mouse model was generated by cloning HIV-1 Nef ORF in CAG-Lox-CAT vector. Transgenic mice lines were crossed with the alpha myosin heavy chain promoter Cre mice to express Nef protein in the heart. Western blot data validated Nef protein expression in the adult mouse heart. Histological evaluation of the effect of Nef protein expression on heart morphology revealed cardiac hypertrophy. Furthermore, heart function analysis shows that Nef expressing mouse lines have depressed heart function. Conclusion: Our study suggests that Nef causes cardiac dysfunction that mimics cardiomyopathy secondary to latent HIV in PLWHA. Further, this mouse model could be useful in studying the effect of Nef protein in different organs or cells using cell-specific Cre recombinase. Moreover, Nef transgenic mice could be useful in generating new therapeutic tools to control comorbidity in PLWHA.