The effects of salinity acclimation on the morphological characteristics and gonadal development of 5-year-old female Japanese eels (Anguilla japonica) were investigated. A 150-day salinity acclimation experiment was conducted to analyze the morphological features and gonadal development of female eels under different salinity conditions. The results showed that, as salinity increased from 0 to 35 over a period of 90 d (with 30 d of acclimation at salinities 15, 25, and 35, respectively), the pectoral circumference (PC), pectoral fin index (PI), gonadosomatic index (GSI), and liver index (LI) of female eels significantly increased (P＜0.05), while the digestive tract index (DTI) and eye diameter index (EI) showed no significant changes with increasing salinity (P＞0.05). Ovarian histological observations revealed a significant increase in oocyte diameter (OD) with increasing salinity (P＜0.05), although the oocytes remained in the oil droplet stage. After continued acclimation to salinity 35, no significant differences were observed in the aforementioned seven indices at 90, 120, and 150 d (P＞0.05). To investigate the effects of flowing water on female eel gonadal development, a flowing water group was acclimated to varying salinities. The results showed that the trends of changes in the seven indicators in the flowing water group of female Japanese eels during the salinity acclimation process were consistent with those of the still water group. Except for OD, the other six indicators in the flowing water group at the same acclimation salinity level showed no significant differences compared with the still water group (P＞0.05). The OD in the flowing water group was significantly larger than that in the still water group at the same salinity (P＜0.05). After 30 d of acclimation at salinity 35, oocytes in the flowing water group entered the primary yolk globule stage, with an oocyte diameter of (161.97±7.46) μm, which was significantly larger than that of the still water group at (140.46±9.36) μm. Determination of gonadotropin levels in the serum of female eels in the still water and flowing water groups revealed that during the entire salinity acclimation period, no significant changes were present in luteinizing hormone (LH) levels at various salinities for both still water and flowing water groups (P＞0.05). The LH levels of female eels in the flowing water group were not significantly different from those in the still water group at the same salinity level (P＞0.05). Salinity acclimation had a significant impact on follicle-stimulating hormone (FSH) levels in both the still-and flow-water groups. After 30 d of acclimation to a salinity of 35, the FSH level in the flowing water group reached (39.66±1.78) U/L, which was significantly higher than that in the still water group (36.97±1.52) U/L (P＜0.05). In summary, salinity is a crucial triggering factor for the gonadal development of female Japanese eels, and the stimulation of flowing water accelerates the early development of oocytes, promoting the synthesis of follicle-stimulating hormones. These findings provide fundamental data and reference information for reproductive biology research on Japanese eels.