Cholinergic neurotransmission affects decision-making, notably through the modulation of perceptual processing in the cortex. In addition, acetylcholine acts on value-based decisions through as yet unknown mechanisms. We found that nicotinic acetylcholine receptors (nAChRs) expressed in the ventral tegmental area (VTA) are involved in the translation of expected uncertainty into motivational value. We developed a multi-armed bandit task for mice with three locations, each associated with a different reward probability. We found that mice lacking the nAChR β2 subunit showed less uncertainty-seeking than their wild-type counterparts. Using model-based analysis, we found that reward uncertainty motivated wild-type mice, but not mice lacking the nAChR β2 subunit. Selective re-expression of the β2 subunit in the VTA was sufficient to restore spontaneous bursting activity in dopamine neurons and uncertainty-seeking. Our results reveal an unanticipated role for subcortical nAChRs in motivation induced by expected uncertainty and provide a parsimonious account for a wealth of behaviors related to nAChRs in the VTA expressing the β2 subunit.