We analysed fatty acids (FAs) composition and the potential functions of FAs in hepatopancreas and testis of freshwater caridean shrimp Macrobrachium nipponense during testis development. The hepatopancreas presented a high unsaturated FA content of about 40%, whereas the testis showed a high level of saturated fatty acids (SFAs) with a decreasing content of 97–86% of total FAs during maturation. Several specific FAs in testis showed significant changes throughout maturation. Among SFAs, C14:0 contributed with a relatively constant high proportion of about 22%, whereas C16:0 and C18:0 contents were also at high levels but showed significant increases during maturation. Representatives of highly unsaturated fatty acids (HUFAs), such as C18:1n‐9, C18:2n‐6, C20:5n‐3 (EPA), C20:4n‐6 (AA) and C22:6n‐3 (DHA) showed significant increases despite their low proportion in the gonad. These results suggest that a high content of HUFAs in hepatopancreas may represent a reservoir for sperm production. The FA C14:0 may provide a particular function. The variations of the FA profile in testis at different developmental stages reveals that C18:1n‐9, C18:2n‐6, C20:5n‐3, C20:4n‐6 and C22:6n‐3 may play important roles in spermiogenesis.