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Astaxanthin is increasingly attracting commercial interest for its application in the nutraceutical and pharmaceutical industries. This study aimed to produce astaxanthin from molasses with our newly mutated strain of <i>Rhodosporidium toruloides</i> G17 and to evaluate biological activities of the produced astaxanthin. To maximize the astaxanthin yield, the response surface methodology was used so as to optimize the culture conditions. A maximum astaxanthin yield of 1262.08 ± 14.58 µg/L was achieved by growing <i>R. toruloides</i> G17 in a molasses-based medium containing 49.39 g/L reducing sugar, 1.00 g/L urea, 4.15 g/L MgSO₄·7H₂O, and 10.05% inoculum ratio. The produced astaxanthin was then purified and studied for its antioxidant and anticancer activities. This compound exhibited 123-fold higher antioxidant activity than α-tocopherol, with an IC₅₀ value of 0.97 ± 0.01 µg/mL. The astaxanthin also showed a potent inhibitory ability against the following three cancer cell lines: HeLa, A549, and MCF7, with IC₅₀ values of 69.07 ± 2.4 µg/mL, 55.60 ± 2.64 µg/mL, and 56.38 ± 4.1 µg/mL, respectively. This study indicates that astaxanthin derived from our newly mutated <i>R. toruloides</i> G17 is a promising anticancer and antioxidant agent for further pharmaceutical applications.