The present study was conducted to determine the 24, 48, 72, and 96-h median lethal concentration (LC50) of acephate and investigate the antioxidant response and acetylcholinesterase (AChE) activities in liver, gill, and spleen of Synechogobius hasta exposed to 0 (control), 5, and 10 mg/L acephate, at the fixed interval time of 24, 48, 72, and 96 h, respectively. LC50 value was 60.83 mg/L at 24 h, 51.36 mg/L at 48 h, 47.07 mg/L at 72 h and 40.13 mg/L at 96 h, respectively. Dismutase (SOD), catalase (CAT), AChE activities, and malondialdehyde (MDA) levels in these tissues for the control remained stable over the exposure period. However, for the two tested groups, tissue-, dose-, and time-dependent responses of these parameters were observed in S. hasta. In general, hepatic SOD and CAT activities were significantly inhibited at 24 h, activated, and increased at 48 h, but again inhibited from 48 to 96 h in fish exposed to the two tested concentrations. Hepatic MDA levels of fish for the two tested concentration peaked at 48 h, significantly higher than the control. Hepatic AChE activity was inhibited at 24 h, peaked at 48 h, and then declined at 72 h for the two tested groups. For gills, the highest SOD and CAT activities for the two tested groups were observed at 48 h, higher than the control. AChE activities for the two tested groups were significantly inhibited at 24 h, but activated at 48 h. At 96 h, AChE activities among the treatments showed no significant differences. Gill MDA levels at 48 h for the tested groups were significantly higher than the control, but showed no significant differences at 24 and 72 h among the treatments. In spleen, SOD and CAT activities at 48 h for the two tested groups were significantly higher than those in the control, but at 96 h the vice versa was true. Spleenic AChE activities and MDA levels for the two tested groups were inhibited at 24 h, activated at 48 h, and then were again inhibited at 72 h. Based on these observations earlier, the results obtained in our study will have important toxicological implications for waterborne acephate pollution and, meantime, provide the basis for the effective risk assessment of acephate in water environment and appropriate safety recommendations for fish. © 2011 Wiley Periodicals, Inc. Environ Toxicol 2013.