Cognitive theories of depression have long posited automatic interpretation biases (AIB) as a central contributor to depressed mood. The current study was first to examine AIB in a clinically defined depressed sample. While assessing AIB using a semantic association paradigm, pupillary reactivity was simultaneously recorded to build insight into the AIB process. A total of 53 individuals (25 depressed and 28 healthy control) completed the Word Sentence Association Paradigm for Depression (WSAP-D) while pupillary reactivity was recorded. Results revealed the depressed group was significantly more likely to endorse negative AIB and less likely to endorse benign AIB compared to healthy controls. The depressed group demonstrated a modest effect size difference indicating they were faster to endorse negative AIB compared to the healthy controls, but did not differ in endorsing benign AIB or in rejecting either valence. Pupillary reactivity was found to differentiate behaviorally defined AIB type from a natural processing condition when counter to theorized, group relevant AIB. The depressed group demonstrated greater initial pupillary constriction during initial presentation of ambiguous information and comparatively less pupillary dilation during and after endorsing a benign AIB. Taken together, the results suggest that theorized negative AIB and lack of benign AIB are characteristic of depression, that greater cognitive effort is required to reject interpretations consistent with theorized biases consistent with reinterpretation processes, and that depressed individuals are less engaged with benign AIB compared to healthy controls, possibly associated with hedonic deficits. Theoretical implications and future directions are discussed.