Background Recent neuroimaging evidence suggests that mindfulness practice may mitigate the biasing influence of prior cognitive and emotional expectations on pain perception. The current study tested this hypothesis using a pain-cueing paradigm, which has reliably been shown to elicit conditioned hypoalgesic and hyperalgesic effects. Specifically, we aimed to investigate whether the instructed use of a mindfulness compared to a suppression strategy differentially modulates the magnitudes of conditioned hypoalgesia and hyperalgesia. Methods Sixty-two healthy non-meditators were assigned to listen to either brief mindfulness or suppression instructions, in between the conditioning and testing phases of a pain-cueing task. Participants provided ratings of anticipatory anxiety, pain intensity and pain unpleasantness throughout the task. They also completed trait and state self-report measures of mindfulness and pain catastrophizing. Results Results indicated that the paradigm was successful in inducing conditioned hyperalgesic and hypoalgesic effects. Importantly, while we found evidence of cue-induced hyperalgesia in both groups, only the suppression group reported cue-induced hypoalgesia. No group differences in pain ratings were found for unconditioned (novel-cued) stimuli. Conclusions These findings provide partial support for recently proposed predictive processing models, which posit that mindfulness may lead to a prioritization of current sensory information over previous expectations. We explore potential explanations for the asymmetrical group differences in conditioned hypoalgesia versus conditioned hyperalgesia, and discuss our results in light of recent neuroimaging insights into the neuropsychological mechanisms of mindfulness and expectancy-driven pain modulation. Significance The current study provides novel insights into the working mechanisms of mindfulness-driven pain modulation. Our data suggest that brief mindfulness training may reduce the influence of prior beliefs and expectations on pain perception. This finding adds to growing evidence suggesting that mindfulness may alleviate pain via neuropsychological mechanisms opposite to those typically observed in conditioning/placebo procedures and other cognitive manipulations. These unique mechanisms underline the potential of mindfulness as an alternative to traditional cognitive pain regulatory strategies.