Congenital heart disease is the leading cause of death in the first year of life. Mutations only in few genes have been linked to some cases of CHD. Thalidomide was used by pregnant women for morning sickness but was removed from the market because it caused severe malformations including CHDs. We used both in silico docking software, and in vitro molecular and biochemical methods to document a novel interaction involving Thalidomide, TBX5, and HAND2. Thalidomide binds readily to TBX5 through amino acids R81, R82, and K226 all implicated in DNA binding. It reduces TBX5 binding to DNA by 40%, and suppresses TBX5 mediated activation of the NPPA and VEGF promoters by 70%. We documented a novel interaction between TBX5 and HAND2, and showed that a p.G202V HAND2 variant associated with CHD and coronary artery diseases found in a large Lebanese family with high consanguinity, drastically inhibited this interaction by 90%. Similarly, thalidomide inhibited the TBX5/HAND2 physical interaction, and the in silico docking revealed that the same amino acids involved in the interaction of TBX5 with DNA are also involved in its binding to HAND2. Our results establish a HAND2/TBX5 pathway implicated in heart development and diseases.