Rationale and objective The aim of this study was to investigate the possible facilitating effect of the partial NMDA receptor agonist D-cycloserine (DCS) on memory consolidation of conditioned sexual responses and to examine the capability of DCS to reduce context-specificity of learning. Methods In a randomized placebo-controlled double-blind trial, 50 healthy females were exposed to a differential conditioning procedure. Two pictures of a male abdomen were used as conditional stimuli (CSs), of which one (the CS+) was followed by the unconditional stimulus (US), a genital vibrotactile stimulus. After the conditioning session on day 1, participants received either 125 mg of DCS or a placebo. The effects of DCS on affect, sexual arousal and US expectancy in response to the CS+ and CS− were examined 24 h after the conditioning procedure.Results A main effect of DCS was found on affect at the first test trials (p = 0.04, η p 2 = 0.09), and a similar non-significant but trend level effect was found for sexual arousal (p = 0.06, η p 2 = 0.07), which appeared to persist over a longer time (p = 0.07, η p 2 = 0.08). Unexpectedly, ratings of positive affect and sexual arousal in response to both the CS+ and the CS− were higher in the DCS condition compared to the control condition, possibly indicating that DCS administration reduced stimulus specificity. Since the results did not show clear evidence for context learning, we were not able to test effects on context-specificity of learning. Conclusion Although largely inconclusive, the results provide tentative support for a facilitating effect of DCS on affect and sexual arousal in response to stimuli that were presented in a sexual conditioning procedure, however, no conclusions can be drawn about effects of DCS on sexual reward learning, since the design and results do not lend themselves to unambiguous interpretation.