Several highly conserved families of guidance molecules, including ephrins, Semaphorins, Netrins, and Slits, play conserved and distinct roles in tissue remodeling during tissue patterning and disease pathogenesis. Primarily, these guidance molecules function as either secreted or surface-bound ligands that interact with their receptors to activate a variety of downstream effects, including cell contractility, migration, adhesion, proliferation, and inflammation. Vascular smooth muscle cells, contractile cells comprising the medial layer of the vessel wall and deriving from the mural population, regulate vascular tone and blood pressure. While capillaries lack a medial layer of vascular smooth muscle, mural-derived pericytes contribute similarly to capillary tone to regulate blood flow in various tissues. Furthermore, pericyte coverage is critical in vascular development, as perturbations disrupt vascular permeability and viability. During cardiovascular disease, smooth muscle cells play a more dynamic role in which suppression of contractile markers, enhanced proliferation, and migration lead to the progression of aberrant vascular remodeling. Since many types of guidance molecules are expressed in vascular smooth muscle and pericytes, these may contribute to blood vessel formation and aberrant remodeling during vascular disease. While vascular development is a large focus of the existing literature, studies emerged to address post-developmental roles for guidance molecules in pathology and are of interest as novel therapeutic targets. In this review, we will discuss the roles of guidance molecules in vascular smooth muscle and pericyte function in development and disease.