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The use of anesthetics has been suggested as a strategy to hamper live fish transport-induced stress. Still, there is insufficient data available on the use of alternative anesthetics to MS-222. This study investigated the use of propofol to mitigate stress in Nile tilapia (Oreochromis niloticus, 143.8 ± 20.9 g and 20.4 ± 0.9 cm) during a 6 h simulated transport. Individuals (n = 7) were divided into three groups: control, 40 mg L−1 MS-222, and 0.8 mg L−1 propofol. A naïve group non-transported was also considered. During the 6 h transport and 24 h after, the response to external stimuli, opercular movements, water quality parameters, behavior, blood hematology and other physiological values, the histopathology of the gills, the quality of the fillet, and oxidative-stress changes in gills, muscle, brain, and liver were evaluated. Propofol increased swimming activity of fish but decreased opercular movements and responses to external stimuli, indicating oscillations of the sedation depth. Water pH and glucose levels increased, while hematocrit (HCT) and lactate decreased in propofol groups at 6 h. At this time-point, MS-222 also induced a decrease in the HCT and lactate levels while increasing cortisol levels. Despite these effects, the stress-related behaviors lessened with anesthetics compared to the control group. After the recovery period, physiological responses normalized in animals from both anesthetic groups, but the control still had high cortisol levels. Overall, propofol is a good alternative for the transportation of this species, showing efficient sedation without compromising health or fillet quality. However, further pharmacodynamics and pharmacokinetics knowledge is required to support its use in aquaculture settings.