Depolarization‐induced suppression of excitation and inhibition (DSE and DSI) appear to be important forms of short‐term retrograde neuronal plasticity involving endocannabinoids (eCB) and the activation of presynaptic cannabinoid CB1 receptors. We report here that CB1‐dependent DSE can be elicited from autaptic cultures of excitatory mouse hippocampal neurones. DSE in autaptic cultures is both more robust and elicited with a more physiologically relevant stimulus than has been thus far reported for conventional hippocampal cultures. An additional requirement for autaptic DSE is filled internal calcium stores. Pharmacological experiments favour a role for 2‐arachidonyl glycerol (2‐AG) rather than arachidonyl ethanolamide (AEA) or noladin ether as the relevant endocannabinoid to elicit DSE. In particular, the latter two compounds fail to reversibly inhibit EPSCs, a quality inconsistent with the role of bona fide eCB mediating DSE. Δ⁹‐Tetrahydrocannabinol (Δ⁹‐THC) fails to inhibit EPSCs, yet readily occludes both DSE and EPSC inhibition by a synthetic CB1 agonist, WIN 55212‐2. With long‐term exposure (∼18 h), Δ⁹‐THC also desensitizes CB1 receptors. Lastly, a functional endocannabinoid transporter is necessary for the expression of DSE.