The aim of this study was to see how chilled sperm quality is affected by ejaculation frequency and its correlation to seminal plasma composition in INRA 180 rams. Five rams were collected at high (HFE) and low frequencies (LFE).For the high frequency, the rams were collected three times on the same day every three days for 18 days. In the low frequency collection, three consecutive ejaculates were collected once a week for four weeks. Ejaculates were collected at 20-minute intervals in either HFE or LFE. Semen characteristics, concentration of total protein, lipid, cholesterol and fructose in seminal plasma were assessed. Semen samples were extended in skim milk-based extender at 15 °C, then evaluated at different storage times (0, 8, and 24 h). Fresh sperm quality parameters, seminal plasma composition, and stored sperm quality were shown to be higher in LFE than in HFE and in the first and second ejaculates than in the third one. After 24 h of storage, sperm quality was correlated to seminal plasma components. In conclusion, the frequency of ejaculation has an effect on the fresh and stored semen quality as well as on seminal plasma composition in INRA 180 rams.