Light is an essential and powerful element to animals. A light-emitting diode (LED) is most efficient in terms of economic benefits. The aim of the present study was to evaluate the effects of LED lighting time on milk production, milk composition, and the immune response of Holstein cows. Forty lactating cows were assigned to four experimental groups: control; natural daylight, treatment; am3-6, pm6-12 and pm6-am6. We found that there was no significant effect on the decrease ratio in milk production among the groups. Milk urea nitrogen (MUN) was significantly decreased in pm6-am6 and pm6-12 than the control. With regard to the hemolytic biochemical analysis, GLU was significantly increased and CRE, T-BIL were significantly decreased in the pm6-12 than the control. IGF-1 levels were significantly increased in pm6-12 compared to other groups. Besides, cortisol was significantly lowered in the pm6-12 than the control, while prolactin, IgA and IgG were not significant among the groups. In addition, catalase and glutathione peroxidase were also significantly increased in pm6-12 than the control. However, antioxidant enzyme activity and superoxide dismutase were not significant among the experimental groups. Therefore, it was concluded that LED lighting time had some impact on blood parameters and immune responses in dairy cows without any changes in milk production.