In sexual organisms, division of the zygote initiates a new life cycle. Although several genes involved in zygote division are known in plants, how the zygote is activated to start embryogenesis has remained elusive. Here, we showed that a mutation in ZYGOTE-ARREST₃ (ZYG₃) in Arabidopsis led to a tight zygote-lethal phenotype. Map-based cloning revealed that ZYG₃ encodes the transfer RNA (tRNA) ligase AtRNL, which is a single-copy gene in the Arabidopsis genome. Expression analyses showed that AtRNL is expressed throughout zygotic embryogenesis, and in meristematic tissues. Using pAtRNL::cAtRNL-sYFP-complemented zyg₃/zyg₃ plants, we showed that AtRNL is localized exclusively in the cytoplasm, suggesting that tRNA splicing occurs primarily in the cytoplasm. Analyses using partially rescued embryos showed that mutation in AtRNL compromised splicing of intron-containing tRNA. Mutations of two tRNA endonuclease genes, SEN₁ and SEN₂, also led to a zygote-lethal phenotype. These results together suggest that tRNA splicing is critical for initiating zygote division in Arabidopsis.