This experiment was conducted to determine the effects of habituation to livestock trailers on stress responses in goats transported for long periods. Intact male Spanish goats (12-month old; BW = 31.6 ± 0.34 kg; N = 168) were separated into two treatment (TRT) groups and maintained on two different paddocks. Concentrate supplement was fed to one group inside two livestock trailers (5.0 × 2.3 m each; habituated group, H), while the other group received the concentrate supplement, but not inside the trailers (non-habituated, NH). After 4 weeks of habituation period, goats were subjected to a 10-h transportation stress in four replicates (n = 21 goats/replicate/TRT). Blood samples were collected by a trained individual by jugular venipuncture into vacutainer tubes before loading (Preload), 20 min after loading (0 h), and at 2-h intervals thereafter (Time) for analysis of stress responses. There was a tendency for a TRT effect (p < 0.1) on tyramine and metanephrine concentrations. Phenylethylamine and 5-methoxytryptamine concentrations were significantly greater (p < 0.05) in the H group compared to the NH group. Both dopamine and 5-methoxytryptamine concentrations decreased (p < 0.05) with transportation time; however, TRT × Time interaction effects were not significant. Habituation to trailers may be beneficial in mood and energy stabilization in goats during long-distance transportation.