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(1) Background: Klotho is a multifunctional protein, but its biophysiological roles during fish early development are unclear. (2) Methods: We carried out anti-sense Klotho morpholino injection and whole-mount in situ hybridization experiments in this study. (3) Results: Our results showed that in the Klotho MO1-injected group, embryos displayed longitudinal shapes and swelling yolks compared to those of the un-injected groups. Their length/width ratio by 9 hpf for the uninfected group is 1 ± 0.1; but for 0.1 mM-, 0.2 mM-, and 0.5 mM-MO-injected are 1.1 ± 0.1, 1.2 ± 0.1, and 1.3 ± 0.1, respectively, suggesting that the klotho knockdown-induced length/width ratio changes are in a dose-dependent manner. Whole-mount in situ hybridization showed that several cell migration-related gene expressions, including goosecoid, floating head, bone morphogenetic protein 4, and even-skipped-liked 1 were affected in the Klotho MO-injected embryos. (4) Conclusions: We suggest that knockdown of klotho leads to cell movement impairment during zebrafish gastrulation. [ABSTRACT FROM AUTHOR]