Supplementation with l-citrulline (Cit) has been shown to improve muscle oxygenation and oxygen uptake kinetics during moderate- to high-intensity cycling in young men. The aim of this study was to test the hypothesis that Cit would improve oxygen uptake kinetics during walking in older and young adults. In a randomized, double-blind study, 26 (15 women, 11 men) adults between the ages of 20–35 years (n = 15) and 64–86 years (n = 11) completed 7-day periods of taking placebo and Cit (6 g/day) in a crossover manner. Participants walked on a treadmill at 40% heart rate reserve while pulmonary oxygen uptake was measured using indirect calorimetry. Net oxygen cost, mean response time (MRT), and the oxygen deficit were calculated before and after each supplement period. There was no significant change (P > 0.05) in net oxygen cost, MRT, or the oxygen deficit after Cit in older adults, while young adults showed a decrease (P = 0.05) in the oxygen deficit after Cit that tended (P = 0.053) to be different than the change after placebo. Sex-stratified analysis revealed that Cit decreased MRT (P = 0.04, Cohen’s d = 0.41) and the oxygen deficit (P < 0.01, Cohen’s d = 0.56) in men with the change after Cit being greater than the change after placebo (MRT: −4.5 ± 2.1 vs. 3.4 ± 2.1 s, P = 0.01; deficit: −0.15 ± 0.05 vs. 0.01 ± 0.05 L, P = 0.02). All oxygen uptake parameters were unchanged (P > 0.05) following Cit and placebo in women. Cit does not alter the oxygen cost of moderate-intensity walking in young or older adults, but Cit improved the rate of rise in oxygen uptake at exercise onset in men.