Hippo signaling pathway is a kinase cascade which plays an important role in organ size control. As the main effectors of the Hippo pathway, transcription coactivators Yap1/Wwtr1 are regulated by the upstream kinase Stk3. Recent studies in mammals have implicated the Hippo pathway in development of different organs and diseases. To further illustrate its roles in vertebrate development, we generated a series of zebrafish mutants targeting stk3, yap1 and wwtr1 genes by CRISPR/Cas9 method and transgenic lines overexpressing Yap1 and Wwtr1. With the analysis of these zebrafish lines, we illustrated the crucial roles of the Hippo pathway in renal cyst formation and scoliosis. The stk3-/- mutant exhibited edema, formation of glomerular cysts and pronephric duct dilation during larval stage. Further experiments showed that the pronephric phenotypes in the stk3-/- mutant were mediated primarily by Wwtr1 but not Yap1. Disruption of wwtr1 but not yap1 gene significantly alleviated the renal phenotypes of the stk3-/- mutant. Overexpression (OE) of Wwtr1 with CMV promoter also induced pronephric phenotypes during larval stage, similar to those of the stk3-/- mutant. Furthermore, adult fishes with Wwtr1 overexpression developed similar phenotypes to those of human polycystic kidney disease (PKD), such as enlarged kidney size, glomerular and renal tubular cyst formation and kidney injury. Using pharmacological approach, we further demonstrated that Stk3-deficient zebrafish could serve as a PKD model for drug development. Overall, our studies revealed roles of Stk3-Wwtr1 in renal cyst formation, which provides novel insight into the involvement of the Hippo signaling pathway in kidney development and function. In addition to kidney development and renal cyst formation, we also observed curved spine (scoliosis) in stk3 f0 mutants. As stk3-/- mutant could not survive to adulthood, we analyzed Yap1 OE and yap1-/- mutant fish. Both of these lines exhibited scoliosis and cilia defects, which implied the involvement of Stk3-Yap1 in scoliosis and the connection between ciliogenesis and body curvature. Together, this study elucidated functions of the Hippo pathway in zebrafish development and established potential zebrafish models for PKD and scoliosis, which may facilitate drug screen and therapy development for these diseases.