Abstract: The present study was carried out to investigate the acute toxicity of TBTO and the effects of its sublethal concentration on brain AChE and gill ATPases activity of tilapia fish, Oreochromis niloticus during acute (3-96 h) and subchronic (7-28 d) exposure periods. The results showed that TBTO is extremely toxic to O. niloticus with 96 h LC50 of 16.3 μg L-1 using static renewal bioassay test. Sublethal concentration of 4.1 μg L-1 (25% of 96 h LC50) of TBTO inhibited brain AChE activity by 24.01 and 24.06% of control at 96 h and 7 d, respectively. Gill Na+, K+-ATPase activity was inhibited by 51.8 and 54.5% at 96 h and 7 d, respectively. The activity of gill Mg2+-ATPase was decreased by 26.9 and 24.28% at 96 h and 21 d, respectively. After 7 d of recovery, AChE and Mg2+-ATPase activites were completely recovered. The activity of Na+, K+-ATPase was partially recovered but the inhibition was still significant. The inhibition percentages were higher in acute exposure than in subchronic exposure in both AChE and Na+, K+-ATPase, but the opposite result was recorded in Mg2+-ATPase. It can be concluded that there is a need for more evaluation and international regulation to minimize the organotin input to aquatic environment.