d-Cycloserine (DCS), a partial NMDA receptor agonist, has been proposed as a cognitive enhancer to facilitate the extinction of drug-related memories. However, it is unknown whether there are individual differences in the efficacy of DCS. Here, we set out to investigate the influence of serotonin transporter (5-HTT) genotype on DCS treatment outcome and the underlying neural mechanism. To that end, we first determined the mRNA levels of several NMDA receptor subunits and observed a reduction in NR1/NR2C receptors in the ventromedial prefrontal cortex and nucleus accumbens of 5-HTT(-/-) compared with 5-HTT(+/+) rats. Based on this finding, we hypothesized a lower sensitivity to DCS in the 5-HTT(-/-) rats. To test this, rats were trained in a cocaine-induced conditioned place preference (CPP) paradigm. A significant extinction of CPP was observed in 5-HTT(+/+) rats receiving 1 mg/kg i.v. DCS, while a similar effect was found in the 5-HTT(-/-) rats only after 5 mg/kg. Following CPP, we tested if DCS were able to reduce FosB/∆FosB protein expression, a molecular switch for cocaine-seeking behaviour. We observed an overall lower number of FosB/∆FosB positive cells in 5-HTT(-/-) ventromedial prefrontal cortex and amygdala and an overall effect of DCS treatment on the number of positive cells in the nucleus accumbens. In conclusion, in this study, we show that the dosing of DCS to facilitate the extinction of cocaine-seeking behaviour is, at least partially, determined by 5-HTT genotype.