The selectivity of (4Z)-2-(4-chloro-3-nitrophenyl)-4-(pyridin-3-ylmethylidene)-1,3-oxazol-5-one (DI) for zipper-interacting protein kinase (ZIPK) was previously described by in silico computational modeling, screening a large panel of kinases, and determining the inhibition efficacy. Our assessment of DI revealed another target, the Rho-associated coiled-coil-containing protein kinase 2 (ROCKII). In vitro studies showed DI to be a competitive inhibitor of ROCKII (Ki, 132 nM with respect to ATP). This finding was supported by in silico molecular surface docking of DI with the ROCKII ATP-binding pocket. Time course analysis of myosin regulatory light chain (LC20) phosphorylation catalyzed by ROCKII in vitro revealed a significant decrease upon treatment with DI. ROCKII signaling was investigated in situ in human coronary artery vascular smooth muscle cells (CASMCs). ROCKII down-regulation using siRNA revealed several potential substrates involved in smooth muscle contraction (e.g., LC20, Par-4, MYPT1) and actin cytoskeletal dynamics (cofilin). The application of DI to CASMCs attenuated LC20, Par-4, LIMK, and cofilin phosphorylations. Notably, cofilin phosphorylation was not significantly decreased with a novel ZIPK selective inhibitor (HS-38). In addition, CASMCs treated with DI underwent cytoskeletal changes that were associated with diminution of cofilin phosphorylation. We conclude that DI is not selective for ZIPK and is a potent inhibitor of ROCKII.