Background: The purpose of the present study was to determine if a polymorphism in the cytochrome P450 CYP1A2 gene impacts performance enhancement with caffeine treatment in recreational cyclists. Furthermore, we explored the impact of this polymorphism on circulating caffeine concentrations during acute exercise. Methods: Twenty subjects (13 males, 7 females) who reported non-to-moderate caffeine usage visited the Exercise Science Laboratory on three occasions. During the first visit, subjects underwent a graded exercise test to determine maximal oxygen consumption (VO2max). A venous blood sample was then obtained for genotyping analysis through polymerase chain reaction and gel electrophoresis. For the next two visits, three pieces of chewing gum (caffeine vs. placebo) were administered in a counterbalanced double blind manner. Subjects remained resting for 10 min and then completed a standard warm-up on a Lode cycle ergometer. Following the warm-up, subjects cycled at 75% VO2max (constant Wattage) for 15 min, rested for 10 min, and then completed a 15-min performance ride. Venous blood samples were collected at baseline (Base), during the warm-up (+25), and immediately before (+50) and after (+65) the performance ride. Serum samples were analyzed for caffeine concentrations through high-performance liquid chromatography. Subjects were grouped based on genotype and performance, and serum caffeine data were analyzed through a repeated-measures analysis of variance (ANOVA). Results: Eleven subjects possessed the AA genotype, while nine subjects were C allele carriers. Independent samples t-tests confirmed that groups were of similar age, body mass, and fitness. The data demonstrated that performance enhancement with caffeine treatment was not evident in this subject population (p ≥ 0.258). Furthermore, the ANOVA demonstrated that ergogenic responses to caffeine did not differ across genotype groups (p ≥ 0.861). Finally, circulating caffeine concentrations were similar across groups with caffeine treatment (p = 0.613). Conclusions: We conclude that a polymorphism in the cytochrome P450 CYP1A2 gene did not impact the ergogenic benefit of caffeine in recreational cyclists. Furthermore, circulating caffeine concentrations did not differ across genotype groups during acute exercise with caffeine treatment.