Using CRISPR/Cas9 genome editing technology, we achieved targeted mutagenesis of the dnd1 gene in freshwater angelfish (Pterophyllum scalare). Subsequent analysis revealed that homozygous mutation of dnd1 resulted in infertility among adult angelfish, characterized by the presence of empty gonad somatic structures without any germ cells. Notably, our observations unveiled a significant male-biased trend in sex development of infertile freshwater angelfish, with the majority exhibiting male secondary sexual characteristics and engaging in normal courtship behaviors. Further investigation indicated a marked upregulation of amh, a key marker for Sertoli cells, in the empty gonad structure of dnd1 KO angelfish, suggesting its pivotal role in male sexual fate determination. Additionally, infertility correlated with a conspicuous increase in visceral fat accumulation, highlighting a potential link between adipose tissue dynamics and reproductive dysfunction. Our research highlights the crucial role of the dnd1 gene in regulating fertility and feminization in freshwater angelfish. Compared to females, infertile male angelfish exhibited notable changes in gut microbiome diversity, but their overall microbial composition closely resembled that of normal males, suggesting that physiological traits remain male-like despite the loss of reproductive function. Core microbiome analysis identified Cetobacterium somerae as the dominant bacterial species, a microbe associated with gut health and antiviral immunity of teleost, in both normal male and infertile male angelfish. Our findings suggest a complex relationship between energy allocation, visceral fat deposition, gonad development and gut microbiota during reproduction in freshwater angelfish.