Human neuroimaging offers a powerful way to connect animal and human research on emotion, with profound implications for psychological science. However, the gulf between animal and human studies remains a formidable obstacle: Human studies typically focus on the cortex and a few subcortical regions such as the amygdala, whereas deeper structures such as the brainstem periaqueductal gray (PAG) play a key role in animal models. Here, we directly assessed the role of PAG in human affect by interleaving physical pain and negative image viewing during a single fMRI session. PAG activity increased in both conditions. We next examined eight independent datasets, half investigating pain and half negative image viewing. We found increased activity in the same portion of the PAG in all eight studies. Taken together, these findings suggest PAG is a key component of human affective responses.