Facial expressions of emotion are thought to have evolved from the development of facial muscles used in sensory regulation and later adapted to express moral judgment. Negative moral judgment includes the expressions of anger, disgust and contempt. Here, we study the hypothesis that these facial expressions of negative moral judgment have further evolved into a facial expression of negation regularly used as a grammatical marker in human language. Specifically, we show that people from different cultures expressing negation use the same facial muscles as those employed to express negative moral judgment. We then show that this nonverbal signal is used as a co-articulator in speech and that, in American Sign Language, it has been grammaticalized as a non-manual marker. Furthermore, this facial expression of negation exhibits the theta oscillation (3–8 Hz) universally seen in syllable and mouthing production in speech and signing. These results provide evidence for the hypothesis that some components of human language have evolved from facial expressions of emotion, and suggest an evolutionary route for the emergence of grammatical markers.