Highly unsaturated fatty acids (HUFA) not only play important physiological functions in human health, they are also closely correlated with individual survival, growth, development, and reproduction in fish. Because marine fish have a low capability or inability to synthetize HUFA, high proportions of HUFA-rich fish oil must be added to compound feedstuff. With the rapid development of aquaculture, fish oil is facing an increasing shortage. Researchers are trying to find a way to improve endogenous HUFA biosynthesis by fish with the intention of increasing the ratio of vegetable oil in formula feeds. The clarification of HUFA biosynthesis will help in achieving this goal. This paper highlights the main factors and mechanisms affecting HUFA biosynthesis, including nutritional factors such as dietary lipid sources, ratio of n-3/n-6 polyunsaturated fatty acids (PUFA) in diets, vitamins, and minerals; environmental factors such as water temperature, salinity, and photoperiod; and transcription factors, hormones, and genetic factors. We hope this information will be a useful reference for future studies on the regulation mechanisms of HUFA biosynthesis in fish.