The study of macromolecular (protein:protein) interactions has significantly enhanced our comprehension of human impairments, such as cardiovascular diseases (CVDs). We previously identified that RhoBTB1 acts as a key regulator of vascular tone by associating with Cullin-3 (CUL3) to control the activity of phosphodiesterase 5 (PDE5) through the ubiquitination-proteasomal pathway. PDE5 selectively hydrolyzes cGMP thus regulating NO-mediated vasodilation. We hypothesized that the PDE5 binding region on RhoBTB1 could be responsible for binding to other unknown partners in RhoBTB1-CUL3 pathway. We truncated RhoBTB1 into its component domains individually (GTPase, BTB1, BTB2, and CT) and in combination (B1B2C, PB1B2, and B2C) and studied their interaction with PDE5 by co-immunoprecipitation (co-IP) in HEK293 cells. These analyses revealed that the B1B2C domain of RhoBTB1 is the minimal region required to bind PDE5. Interestingly, the CT region is essential as the truncations lacking CT could not participate in PDE5 binding and its subsequent proteasomal degradation. The GTPase and Proline-rich (P) domains were dispensable for binding of RhoBTB1 to PDE5. We identified that Pro 353 and Ser 363 are key amino acid residues in the B1B2C region involved in CUL3 binding to RhoBTB1. Mutation of these amino acids resulted in hampered binding to CUL3. The mutants failed to degrade PDE5 despite their preserved binding to PDE5. We employed ascorbate peroxidase (APEX2) labeling using a B1B2C-APEX2 fusion construct as a bait to identify unknown RhoBTB1 binding partners. As a contemporaneous screen, we used B1B2-APEX2 since it does not bind to PDE5, and therefore is not expected to bind to those unknown partners. We “filtered” the captured proteome for its interaction with B1B2C better than B1B2 (without the CT). SET Domain Containing 2 (SETD2) was among several B1B2C binding partners identified and validated. SETD2 and RhoBTB1 directly interacted, and the level of SETD2 increased in response to RhoBTB1-inhibition with siRNA, inhibition of proteasomal machinery either by pharmacological means or CUL3 editing. This suggests that SETD2 is regulated by the RhoBTB1-CUL3 axis. Future studies may pave a way to determine if SETD2 is important for cardiovascular function.