The zebrafish sensory posterior lateral line (pLL) has become an attractive model for studying collective cell migration and cell morphogenesis. Recent studies have indicated that chemokine, Wnt/β-catenin, Fgf, and Delta-Notch signaling pathways participate in regulating pLL development. However, it remains unclear whether TGFβ signaling pathway is involved in pLL development. Here we report a critical role of TGFβ1 in regulating morphogenesis of the pLL primordium (pLLP). The tgfβ1a gene is abundantly expressed in the lateral line primordium. Knockdown or knockout of tgfβ1a leads to a reduction of neuromast number, an increase of inter-neuromast distance, and a reduced number of hair cells. The aberrant morphogenesis in embryos depleted of tgfβ1a correlates with the reduced expression of atoh1a, deltaA, and n-cadherin/cdh2, which are known important regulators of the pLLP morphogenesis. Like tgfβ1a depletion, knockdown of smad5 that expresses in the pLLP, affects pLLP development whereas overexpression of a constitutive active Smad5 isoform rescues the defects in embryos depleted of tgfβ1a, indicating that Smad5 mediates tgfβ1a function in pLLP development. Therefore, TGFβ/Smad5 signaling plays an important role in the zebrafish lateral line formation.