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Abstract Phosphodiesterase‐5 (PDE5) is responsible for regulating the concentration of the second messenger molecule cGMP by hydrolyzing it into 5′‐GMP. PDE5 is implicated in erectile dysfunction and cardiovascular diseases. The substrate binding site in the catalytic domain of PDE5 is surrounded by several dynamic structural motifs (including the α14 helix, M‐loop, and H‐loop) that are known to switch between inactive and active conformational states via currently unresolved structural intermediates. We evaluated the conformational dynamics of these structural motifs in the apo state and upon binding of an allosteric inhibitor (evodiamine) oravanafil, a competitive inhibitor. We employed enhanced sampling‐based replica exchange solute scaling (REST2) method, principal component analysis (PCA), time‐lagged independent component analysis (tICA), molecular dynamics (MD) simulations, and well‐tempered metadynamics simulations to probe the conformational changes in these structural motifs. Our results support a regulatory mechanism for PDE5, where the α14 helix alternates between an inward (lower activity) conformation and an outward (higher activity) conformation that is accompanied by the folding/unfolding of the α8′ and α8″ helices of the H‐loop. When the allosteric inhibitor evodiamine is bound to PDE5, the inward (inactive) state of the α14 helix is preferred, thus preventing substrate access to the catalytic site. In contrast, competitive inhibitors of PDE5 block catalysis by occupying the active site accompanied by stabilization of the outward conformation of the α14 helix. Defining the conformational dynamics underlying regulation of PDE5 activation will be helpful in rational design of next‐generation small molecules modulators of PDE5 activity.