ARNT factors are a duster of bHLH-PAS factors that heterodimerize with other specific bHLH-PAS factors to mediate a wide range of biological responses. Previously, we obtained a truncated form of ARNT2-like factor, ARNT2A, from zebrafish, which encompasses the basic-helix-loop-helix and PAS A/B domains, but lacks a transactivation domain at its carboxyl end. Herein, we report another truncated ARNT2-like factor, ARNT2X, in zebrafish, which differs from ARNT2A at its N-terminal region. In cultured ZLE cells, transiently expressed ARNT2X and ARNT2A inhibited 2,3,7,8-TCDD-activated cyp1a1 transcription with different efficiencies. In the developing embryo, arnt2X mRNA was consistently expressed in the retinal and neural tube regions until the hatching stages, but it exhibited a more specific pattern at larval stages, including expression in the brain, eyes, hypothalamus, pharyngeal skeleton, heart, liver, pronephros duct, pectoral fin, and epithelial cells of the swim bladder. In contrast, arnt2A transcription diminished after hatching. Microinjecting a recombinant arnt2X-expression vector into fertilized eggs before cleavage stages caused severe defects in brain, eyes, pectoral fin, heart, and gut development. This suggests that the ARNT-mediated signal transduction pathways play important roles in fish tissue development.