The objective of this study was to evaluate the effects of natural astaxanthin (
ASTA
) from
Haematococcus pluvialis
on production performance, egg quality, antioxidant enzyme activity, free radical scavenging ability, and gene expression of antioxidant enzymes in laying hens. Nongda No. 3 laying hens (n = 450) were randomly allocated to 1 of 5 dietary treatments. Each treatment had 6 replicates of 15 hens each. All birds were assigned to a corn-soybean meal-based diet containing 0, 20, 40, 80, or 160 mg/kg ASTA for 4 wk. With increasing dietary ASTA, no significant effects were observed on egg weight, feed consumption, feed efficiency, laying rate, Haugh unit, or eggshell strength. Yolk color darkened linearly with increasing dose of ASTA (
P
< 0.05). Glutathione peroxidase activity was improved in the kidney with dietary ASTA at levels of 40 mg/kg. Total superoxide dismutase (
SOD
) was significantly increased in the liver, kidney, and plasma with dietary ASTA supplementation at 40 mg/kg. With increasing dietary ASTA, the scavenging abilities of hydroxyl radicals and superoxide anions were linearly increased (
P
< 0.05), and the malondialdehyde content decreased linearly (
P
< 0.05). Compared with the control group, mRNA expression of Cu-Zn SOD (
SOD1
), Mn SOD (
SOD2
), and nuclear factor E2-related factor 2 (
NRF2
) in the liver and kidney was significantly increased in the 40 mg/kg ASTA group (
P
< 0.05). The level of
GPX4
mRNA in the liver and kidney was significantly increased with ASTA supplementation at 40 and 80 mg/kg (
P
< 0.05). The results demonstrate that dietary ASTA improves free radical scavenging ability and antioxidant enzyme activity, which may be related in part to the upregulated mRNA expression of genes encoding antioxidant enzymes and
NRF2
.