<p>Digestive enzymes of fish are critical to food digestion at the larval stage, but convincing evidence proving the function and necessity of the associated digestive enzymes remains lacking. In this study, we generated the trypsin (<i>try</i>) gene and amylase (<i>amy</i>) gene in the Japanese medaka (<i>Oryzias latipes</i>) using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) for the first time. <i>try</i> deletion significantly decreased the expression of <i>try</i> and digestive capacity in <i>try</i>^−/− medaka larvae; after 8.5 h of digestion, incompletely digested brine shrimp was observed in the digestive tract at 4 and 15 d post-hatching (dph) of <i>try</i>^−/− medaka larvae. Furthermore, the height of intestinal villi and total body length decreased significantly within 15-dph <i>try</i>^−/− medaka larvae. However, <i>amy</i> deletion did not influence the digestion of medaka larvae at 4 dph. Only a small amount of incompletely digested brine shrimp was observed in 15-dph <i>amy</i>^−/− medaka larvae. Further analysis of the growth, nitrogen metabolism, and intestinal microbes of <i>try</i>^−/− adult medaka showed that the body length and weight of adult medaka decreased significantly, while the activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the blood significantly increased. Pathological observation of the liver and intestinal tissues showed that <i>try</i> knockout resulted in vacuolar degeneration of liver cells, thinning of the intestinal wall, sparse arrangement of villi, and lower villi height. High-throughput 16S ribosomal RNA (rRNA) sequencing revealed that <i>try</i> knockout reduced the diversity of intestinal microbes. These findings demonstrated that <i>try</i> was indispensable for medaka larvae because it continuously affects their growth, nitrogen metabolism, and intestinal development.</p>