Today’s available therapies to treat patients infected with human immunodeficiency virus (HIV) aim at preventing viral replication and transmission but fail to eliminate the virus. Although transplantation of an allogeneic CCR5Δ32 homozygous stem cell grafts provided a cure for three patients, this approach is not considered a general therapeutic strategy because of potential severe side effects. Conversely, genome editing to disrupt the CCR5 locus that encodes the major HIV coreceptor was shown to confer resistance to R5-tropic HIV strains on the cellular level. Here, we present a clinically relevant and highly efficient approach to produce HIV-1 resistant CD4+ T cells. After transferring mRNA coding for CCR5-targeting TALEN into CD4+ T cells by electroporation, up to 89% of CCR5 alleles were disrupted. Genotyping confirmed genetic stability of the edited cell product and off-target analyses established absence of relevant mutagenic events. When challenging these edited T cells with R5-tropic HIV, we observed protection in a dose-dependent manner. Functional assessments revealed no significant differences between edited and control CD4+ T cells in terms of proliferation capacity and their ability to secrete cytokines upon exogenous stimuli. Overall, we successfully engineered HIV-resistant CD4+ T cells under clinically relevant conditions, paving the way for clinical translation.