Cis-regulatory elements (CRE) are critical for coordinating gene expression programs that dictate cell-specific differentiation and homeostasis. Recently developed self-transcribing active regulatory region sequencing (STARR-Seq) has allowed for genome-wide annotation of functional CREs. Despite this, STARR-Seq assays are only employed in cell lines, in part, due to difficulties in delivering reporter constructs. Herein, we implemented and validated a STARR-Seq–based screen in human CD4+ T cells using a non-integrating lentiviral transduction system. Lenti-STARR-Seq is the first example of a genome-wide assay of CRE function in human primary cells, identifying thousands of functional enhancers and negative regulatory elements (NREs) in human CD4+ T cells. We find an unexpected difference in nucleosome organization between enhancers and NRE: enhancers are located between nucleosomes, whereas NRE are occupied by nucleosomes in their endogenous locations. We also describe chromatin modification, eRNA production, and transcription factor binding at both enhancers and NREs. Our findings support the idea of silencer repurposing as enhancers in alternate cell types. Collectively, these data suggest that Lenti-STARR-Seq is a successful approach for CRE screening in primary human cell types, and provides an atlas of functional CREs in human CD4+ T cells.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12864-023-09288-3.