Ca2+ sensitivity of arterial contractility is governed by regulating myosin phosphatase activity in response to agonist stimuli. CPI‐17, a myosin phosphatase inhibitor phosphoprotein, is phosphorylated concomitantly with agonist‐induced contractile Ca2+ sensitization in mammalian artery. CPI‐17 has not been detected in chicken artery, but is readily detectable in pigeon artery. To evaluate a role of CPI‐17, we compared contractility of the arteries of ‘CPI‐17‐deficient’ chicken with those of CPI‐17‐rich rabbit and pigeon, and studied the effect of CPI‐17‐reconstitution in chicken artery. Other major regulatory/contractile proteins for Ca2+ sensitization are expressed in both chicken and rabbit arteries. Agonists, such as an α1‐agonist and endothelin‐1, produced significant contraction in arteries of all species under physiological Ca2+‐containing conditions. Depletion of Ca2+ abolished these contractions in chicken but partially inhibited them in rabbit and pigeon arteries. Unlike CPI‐17‐rich tissues, chicken arteries exerted little Ca2+ sensitization in response to α1‐agonist or endothelin‐1. GTPγS produced a slight Ca2+ sensitizing effect in chicken artery, but this was significantly smaller compared with CPI‐17‐rich tissues. A PKC activator (PDBu) did not generate but rather reduced a contraction in both intact and α‐toxin‐permeabilized chicken artery in contrast to a large contraction in CPI‐17‐rich arteries. Myosin light chain phosphorylation was reduced by PDBu in chicken but elevated in rabbit artery. Addition of recombinant CPI‐17 into β‐escin‐permeabilized chicken artery restored PDBu‐induced and enhanced GTPγS‐induced Ca2+ sensitization. Thus, CPI‐17 is essential for G protein/PKC‐mediated Ca2+ sensitization in smooth muscle.