The endocannabinoid (eCB) system modulates several phenomena related to addictive behaviors, and drug-induced changes in eCB signaling have been postulated to be important mediators of physiological and pathological reward-related synaptic plasticity. Here, we studied eCB-mediated long-term depression (eCB-LTD) in the dorsolateral striatum, a brain region critical for acquisition of habitual and automatic behavior. We report that nicotine differentially affects ex vivo eCB signaling depending on previous exposure in vivo. In the nicotine-naïve brain, nicotine facilitates eCB-signaling and LTD, whereas tolerance develops to this facilitating effect after subchronic exposure in vivo. In the end, a progressive impairment of eCB-induced LTD is established after protracted withdrawal from nicotine. Endocannabinoid-LTD is reinstated 6 months after the last drug injection, but a brief period of nicotine re-exposure is sufficient to yet again impair eCB-signaling. LTD induced by the cannabinoid 1 receptor agonist WIN55,212–2 is not affected, suggesting that nicotine modulates eCB production or release. Nicotine-induced facilitation of eCB-LTD is occluded by the dopamine D2 receptor agonist quinpirole, and by the muscarinic acetylcholine receptor antagonist scopolamine. In addition, the same compounds restore eCB-LTD during protracted withdrawal. Nicotine may thus modulate eCB-signaling by affecting dopaminergic and cholinergic neurotransmission in a long-lasting manner. Overall, the data presented here suggest that nicotine facilitates eCB-LTD in the initial phase, which putatively could promote neurophysiological and behavioral adaptations to the drug. Protracted withdrawal, however, impairs eCB-LTD, which may influence or affect the ability to maintain cessation.