Propranolol and atenolol, current therapies for problematic infantile hemangioma (IH), are composed of R(+) and S(-) enantiomers: the R(+) enantiomer is largely devoid of β-blocker activity. We investigated the effect of R(+) enantiomers of propranolol and atenolol on the formation of IH-like blood vessels from hemangioma stem cells (HemSC) in a murine xenograft model. Both R(+) enantiomers inhibited HemSC vessel formation in vivo. In vitro, similar to R(+) propranolol, both atenolol and its R(+) enantiomer inhibited HemSC to endothelial differentiation. As our previous work implicated the transcription factor SRY(Sex Determining Region Y)-Box Transcription Factor-18 (SOX18) in propranolol-mediated inhibition of HemSC to endothelial differentiation, we tested in parallel a known SOX18 small molecule inhibitor (Sm4) and show that this compound inhibited HemSC vessel formation in vivo with a similar efficacy as the R(+) enantiomers. We next examined how R(+) propranolol alters SOX18 transcriptional activity. Using a suite of biochemical, biophysical and quantitative molecular imaging assays we show that R(+) propranolol directly interferes with SOX18 target gene trans-activation, disrupts SOX18-chromatin binding dynamics and reduced SOX18 dimer formation. We suggest the R(+) enantiomers of widely used β-blockers could be repurposed to increase efficiency of current IH treatment and lower adverse associated side effects.