Wilm's tumor 1 interacting protein (Wtip) was identified as an interacting partner of Wilm's tumor protein (WT1) in a yeast two-hybrid screen. WT1 is expressed in the proepicardial organ (PE) of the heart, and mouse and zebrafish wt1 knockout models appear to lack the PE. Wtip's role in the heart remains unexplored. In the present study, we demonstrate that wtip expression is identical in wt1a‑, tcf21‑, and tbx18‑positive PE cells, and that Wtip protein localizes to the basal body of PE cells. We present the first genetic evidence that Wtip signaling in conjunction with WT1 is essential for PE specification in the zebrafish heart. By overexpressing wtip mRNA, we observed ectopic expression of PE markers in the cardiac and pharyngeal arch regions. Furthermore, wtip knockdown embryos showed perturbed cardiac looping and lacked the atrioventricular (AV) boundary. However, the chamber‑specific markers amhc and vmhc were unaffected. Interestingly, knockdown of wtip disrupts early left‑right (LR) asymmetry. Our studies uncover new roles for Wtip regulating PE cell specification and early LR asymmetry, and suggest that the PE may exert non‑autonomous effects on heart looping and AV morphogenesis. The presence of cilia in the PE, and localization of Wtip in the basal body of ciliated cells, raises the possibility of cilia-mediated PE signaling in the embryonic heart.