The transactivating response element (TAR) of human immunodeficiency virus 1 (HIV-1) is essential for promoter transactivation by the viral transactivator of transcription (Tat). The Tat-TAR interaction thereby recruits active positive transcription elongation factor b (P-TEFb) from its inactive, 7SK/HEXIM1-bound form, leading to efficient viral transcription. Here, we show that the 7SK RNA-associating chromatin regulator HMGA1 can specifically bind to the HIV-1 TAR element and that 7SK RNA can thereby compete with TAR. The HMGA1-binding interface of TAR is located within the binding site for Tat and other cellular activators, and we further provide evidence for competition between HMGA1 and Tat for TARbinding. HMGA1 negatively influences the expression of a HIV-1 promoter-driven reporter in a TAR-dependent manner, both in the presence and in the absence of Tat. The overexpression of the HMGA1-binding substructure of 7SK RNA results in a TAR-dependent gain of HIV-1 promoter activity similar to the effect of the shRNA-mediated knockdown of HMGA1. Our results support a model in which the HMGA1/TAR interaction prevents the binding of transcription-activating cellular co-factors and Tat, subsequently leading to reduced HIV-1 transcription.