A major subpopulation of 5-hydroxytryptamine (5-HT) neurons expresses the vesicular glutamate transporter 3 (VGLUT3) allowing the co-release of glutamate. Previous evidence has implicated VGLUT3 in 5-HT neurons in mechanisms of anxiety and reward. Here we examined mice with a genetic loss of VGLUT3 targeted to 5-HT neurons (VGLUT3 cKO5-HT) and littermate controls in a battery of behavioural tests, including paradigms assessing levels of anxiety and learning with appetitive rewards. Compared to littermate controls, VGLUT3 cKO5-HT mice displayed no evidence of altered anxiety-like behaviour in the elevated plus maze, light/dark box, marble burying and social interaction tests. However, VGLUT3 cKO5-HT mice showed reduced preference for low (but not high) sucrose-containing solution and reduced correct responses in an appetitively motivated spatial reference memory task. Similarly, in an appetitively motivated operant task, VGLUT3 cKO5-HT mice displayed evidence of reduced responding to cues associated with reward. These effects appeared specific in that VGLUT3 cKO5-HT mice did not differ from controls in terms of home cage food consumption, performance in a spatial novelty preference test, as well as contextual and cued fear memory tests. These findings support a role for VGLUT3 in 5-HT neurons in some aspects of learning, here in association with learning for reward, although not anxiety-like behaviour.