Transcription can pose a threat to genomic stability through the formation of R-loops that obstruct the progression of replication forks. R-loops are three-stranded nucleic acid structures formed by an RNA-DNA hybrid with a displaced non-template DNA strand. We developed RDProx to identify proteins that regulate R-loops in human cells. RDProx relies on the expression of the hybrid-binding domain (HBD) of Ribonuclease H1 (RNaseH1) fused to the ascorbate peroxidase (APEX2) and permits mapping of the R-loop proximal proteome using quantitative mass spectrometry. We implicate different cellular proteins in R-loop regulation and identify a role of the tumor suppressor DEAD box protein 41 (DDX41) in opposing R-loop-dependent genomic instability. Depletion of DDX41 resulted in replication stress, double strand breaks and inflammatory signaling. DDX41 opposes the accumulation of R-loops by unwinding RNA-DNA hybrids at gene promoters and its loss leads to upregulation of TGFβ and NOTCH signaling genes. Germline loss-of-function mutations in DDX41 lead to predisposition to acute myeloid leukemia (AML) in adulthood. We propose that accumulation of R-loops at CpG island promoters, altered TGFβ and NOTCH signaling, and inflammatory response contribute to the development of familial AML with mutated DDX41.