Rationale In the present study, it was investigated whether smokers can acquire a behavioural approach bias through Pavlovian conditioning. Objective More specifically, it was tested whether pairing neutral stimuli with either smoking availability or unavailability would lead to both differential urge responding to these stimuli and a corresponding shift in approach bias. Materials and methods Thirty-nine low-dependent smokers performed a stimulus-response compatibility (SRC) task with which one can determine an approach bias. Next, participants received a conditioning session in which one cue (either a blue or yellow background screen colour) was paired with the opportunity to smoke (CS+) and another cue was paired with the absence of the opportunity to smoke (CS-). After conditioning, all participants again performed the SRC task. Results Evidence for the conditioning of an approach bias but not smoking urges was found. This effect, although, was only apparent when smokers had been prompted to determine the contingency between the cues and smoking outcome. Conclusions It is concluded that one can differentially condition an approach bias in low-dependent smokers.