Several studies have shown that the exposure of semen to red light improves sperm quality and fertilizing ability, which could improve the efficiency of assisted reproductive techniques with irradiated semen. However, despite being considered as possible sources of variation, the effects of the color of the container (straws) or the medium have not yet been evaluated. In this study, 13 ejaculates from different stallions were split into equal fractions, diluted either with Kenney or Equiplus extender, and subsequently packed into straws of five different colors. After storage at 4 °C for 24 h, the sperm were irradiated and different variables, including sperm motility, plasma membrane integrity, and mitochondrial membrane potential, were evaluated. Our results confirm that irradiation increases some motion characteristics and mitochondrial membrane potential without affecting sperm viability and demonstrate that the effects depend on the color of the straw and the extender used. Previous research has determined that irradiation of mammalian sperm with red light increases motility, mitochondrial activity, and fertilization capacity. In spite of this, no study has considered the potential influence of the color of the straw and the extender used. Therefore, this study tests the hypothesis that the response of mammalian sperm to red light is influenced by the color of the straw and the turbidity/composition of the extender. Using the horse as a model, 13 ejaculates from 13 stallions were split into two equal fractions, diluted with Kenney or Equiplus extender, and stored at 4 °C for 24 h. Thereafter, each diluted fraction was split into five equal aliquots and subsequently packed into 0.5-mL straws of red, blue, yellow, white, or transparent color. Straws were either nonirradiated (control) or irradiated with a light-dark-light pattern of 3-3-3 (i.e., light: 3 min, dark: 3 min; light: 3 min) prior to evaluating sperm motility, acrosome and plasma membrane integrity, mitochondrial membrane potential, and intracellular ROS and calcium levels. Our results showed that irradiation increased some motion variables, mitochondrial membrane potential, and intracellular ROS without affecting the integrities of the plasma membrane and acrosome. Remarkably, the extent of those changes varied with the color of the straw and the extender used; the effects of irradiation were more apparent when sperm were diluted with Equiplus extender and packed into red-colored straws or when samples were diluted with Kenney extender and packed into transparent straws. As the increase in sperm motility and intracellular ROS levels was parallel to that of mitochondrial activity, we suggest that the impact of red light on sperm function relies upon the specific rates of energy provided to the mitochondria, which, in turn, vary with the color of the straw and the turbidity/composition of the extender.