Abstract <p>The acute toxicity of the herbicide penoxsulam and its ability to induce behavioural changes in freshwater teleost, <i>Labeo rohita</i> was investigated under laboratory conditions. The obtained 96 h median lethal concentration value (2.33 mg/L) of penoxsulam suggested that this herbicide is more toxic to <i>L. rohita</i> than other previously studied species. All studied behavioural endpoints clearly indicated that penoxsulam is having ability to alter the behaviour of <i>L. rohita</i>, which in turn can change the entire physiological aspects of fish and so drastically affect its survival ability. Group forming ability of fish decreases with increasing concentration of penoxsulam. The ability to maintain equilibrium in water also decreases along with the increasing concentration of the herbicide. The buccal movement per minute also increases with the increasing concentration, but the number of burst swimming per minute increases initially but then decreases with increasing concentration of penoxsulam. Thus the acute toxicity bioassay and behavioural toxicity test indicated that herbicide penoxsulam is toxic to <i>L. rohita</i> and the toxicity is dependent on the concentration and duration of exposure of herbicide. Thus excessive and improper use of penoxsulam in agricultural fields must be avoided.</p>