Background Chronic exertional compartment syndrome (CECS) is a condition in which muscle tissue expands against the surrounding fascia during activity and is compressed along with the nerves and blood vessels within the muscle compartment, leading to abnormally high intracompartmental pressure (ICP) and debilitating pain. Treatment typically includes fasciotomy, which results in significant levels of CECS recurrence; however, botulinum toxin A (BoNT-A) injection has recently been seen to decrease both ICP and pain through an unknown mechanism with little to no recurrence. Methods In this study, PyRosetta was used to model the probability of docking interaction between BoNT-A light or heavy chain and enzymes cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), prostaglandin E2 receptor 4 (EP4), endothelial nitric oxide synthase (eNOS), and inducible nitric oxide synthase (iNOS). Subsequently, myoblasts (HSkM) were treated with BoNT-A and assayed based on PyRosetta predicted interactions. Results The EP4/BoNT-A heavy chain combination was found to have reaction scores most comparable to known interactions of BoNT-A in neural cells. In accordance with this finding, levels of cyclic adenosine monophosphate (cAMP), a downstream effector of EP4, consistently increased in myoblasts treated with BoNT-A. Conclusion Altogether, these data uncover an area of future research in determining the interaction of BoNT-A and the EP4 pathway in muscle cells, as well as the implications this may have on CECS and its treatment.