Roosters homozygous for the rose comb allele (R/R) are subfertile. In previous research, these subfertile roosters were characterized by an in vitro sperm penetration assay as having limited sperm motility. The objectives in the present study were to characterize sperm motility by computer-assisted sperm motion analysis and to account for a mechanism underlying poor sperm motility. Percentages of motile sperm differed between subfertile males and fertile controls (r/r) by 29% (p < 0.001). The concentration of intracellular ATP in sperm form subfertile roosters was less than in that from fertile controls (p < 0.001). The genotypic difference is sperm motility, as measured with the sperm penetration assay, was maintained when ATP production was dependent on anaerobic glycolysis (p < 0.001). In this case, sperm were incubated with exogenous glucose and cyanide. Consequently, we could not attribute the genotypic difference in sperm mobility to mitochondrial respiration. In contrast, glucose transport, as measured by the uptake of [1,2-3H]-2-deoxy-D-glucose, was reduced in sperm from subfertile roosters (p < 0.001). Neither hexokinase nor glyceraldehyde-3-phosphate dehydrogenase activity differed between genotypes (p > 0.05). Likewise, lactate dehydrogenase activity did not differ between genotypes (p > 0.05). As evidenced by creatine kinase activity and dynein ATPase activity, neither the potential for energy transfer nor utilization within the axoneme differed between genotypes (p > 0.05). Therefore, we attribute the subfertility of roosters homozygous for the rose comb allele to decreased spermatozoal glucose transport.