The main reason for abnormal development of cloned animals or embryos, and inefficient animal cloning, is a poor understanding of the reprogramming mechanism. To better comprehend reprogramming and subsequent generation of pluripotent stem cells, we must investigate factors related to reprogramming of somatic cells as nuclear donors. As we know, fam60al (family with sequence similarity 60, member A, like) is a coding gene only found in zebrafish and frog (Xenopus laevis) among vertebrates. However, until now, its functions have remained unknown. Here, we generated a zebrafish fam60al(-/-) mutant line using transcription activator-like effector nucleases (TALENs), and found that both nanog and klf4b expression significantly decreased while myca expression significantly increased in fam60al(-/-) mutant embryos. Concurrently, we also uncovered that in developmentally arrested embryos of somatic cell nuclear transfer, nanog, klf4b and myca expression was down-regulated, accompanying a decrease of fam60al expression. Interestingly, we identified a long noncoding RNA (lncRNA) of fam60al, named fam60al-AS, which negatively regulated fam60al by forming double-stranded RNA (dsRNA). RNase protection assay and real-time PCR confirmed these findings. Taken together, these results suggest that fam60al is a novel factor related to the reprogramming of somatic cell nuclear transfer in zebrafish, which is regulated by its reverse lncRNA.