Myosin phosphatase-Rho interacting protein (p116Rip ) was originally found as a RhoA-binding protein. Subsequent studies by us and others revealed that p116Rip facilitates myosin light chain phosphatase (MLCP) activity through direct and indirect manners. However, it is unclear how p116Rip regulates myosin phosphatase activity in cells. To elucidate the role of p116Rip in cellular contractile processes, we suppressed the expression of p116Rip by RNA interference in human airway smooth muscle cells (HASMCs). We found that knockdown of p116Rip in HASMCs led to increased di-phosphorylated MLC (pMLC), that is phosphorylation at both Ser19 and Thr18. This was because of a change in the interaction between MLCP and myosin, but not an alteration of RhoA/ROCK signaling. Attenuation of Zipper-interacting protein kinase (ZIPK) abolished the increase in di-pMLC, suggesting that ZIPK is involved in this process. Moreover, suppression of p116Rip expression in HASMCs substantially increased the histamine-induced collagen gel contraction. We also found that expression of the p116Rip was decreased in the airway smooth muscle tissue from asthmatic patients compared with that from non-asthmatic patients, suggesting a potential role of p116Rip expression in asthma pathogenesis.