The nutrient content and in vitro digestibilty of rejected red seaweed (Eucheuma cottonii) from three different origin locations (regions), Alor, Ambon, and Rote was studied. Rejected seaweed Eucheuma cottoni was obtained from an agar factory located in west kupang subdistrict, Kupang regency, NTT province, Indonesia. Proximate analysis, fiber analysis (NDF, ADF, cellulose, hemicellulose, lignin, silica), mineral analysis (Ca, Mg, Fe, and S) as well as in vitro digestibility (dry matter, organic matter, crude protein, crude fat, crude fiber, NDF, ADF, and lignin) had been studied. Seaweed’s growth location influenced chemical composition and digestibility. In vitro digestibility of crude protein, crude fat, crude fiber, NDF, ADF, and lignin of rejected Eucheuma cottoni from Ambon was higher (p<0.01) than that of rejected Eucheuma cottoni from Alor and Rote. Crude protein and nitrogen free extract content of rejected Eucheuma cottoni from Ambon were also high compared with that of rejected Eucheuma cottoni from Alor and Rote. It is concluded that rejected Eucheuma cottoni from Ambon is better to be used as carbohydrate source feed in calf starter.