The current work aim is to evaluate the oral acute and sub-chronic toxicity of Zeaxanthin-enriched fraction from Dunaliella salina (D. salina). In the acute study, mice were orally administered 0.625, 1.25, 2.5 and 5 g/kg of the Zeaxanthin-enriched fraction as a single dose and observed for two weeks. In the chronic study, Wistar rats were orally administered 250 and 500 mg/kg of the Zeaxanthin-enriched fraction for three months. Complete blood picture (CPC), kidney functions (urea, creatinine, and albumin), liver function enzyme activities (alanine and aspartate amino transferases, alkaline phosphatase, bilirubin, and albumin), and blood glucose level were assessed. Finally, liver, kidney, and heart histopathological investigations were performed. The HPLC analysis showed that hexane/ethanol (2:1, V/V) crude extract of D. salina contains zeaxanthin (‎8.469mg/g crude extract), while the Zeaxanthin-enriched fraction contained zeaxanthin ‎‎(14.301 mg/g rich fraction).In acute toxicity, no toxicologically relevant findings were noted after 24 h of the Zeaxanthin-enriched fraction (0.625, 1.25, 2.5 and 5 g/kg) compared with the control group mice. In the chronic study, both sexes of rats treated for three consecutive months with a daily dose of 250 and 500 mg /kg of the Zeaxanthin-enriched fraction did not show any sign of toxicity (no mortality, no patches of yellow color appearance, no hair, no diarrhea loss and no abnormalities on behavior). The Zeaxanthin-enriched fraction also did not change CPC, kidney functions, liver enzyme activities, and blood glucose level, compared to their relative normal values. The histopathological study, showed normal kidney, liver, and heart in the Zeaxanthin-enriched fraction groups. The Zeaxanthin-enriched fraction LD50 is greater than 5000 mg/kg and is safe at a dose of 500 mg /kg in a chronic study.