Background Limited health literacy is a risk factor for poor outcomes in numerous health care settings. Little is known about the impact of instructional modality and health literacy on adherence to emergency department (ED) discharge instructions. Purpose To examine the impact of instructional modality on 72-hour antibiotic retrieval among ED patients prescribed outpatient antibiotics for infections. Methods English-speaking ED patients diagnosed as having acute infections and prescribed outpatient antibiotics were randomized to standard discharge instructions, standard instructions plus text-messaged instructions, or standard instructions plus voicemailed instructions targeting ED prescriptions. Health literacy was determined by validated instrument. Seventy-two-hour antibiotic retrieval, 30-day report of prescription completion, and discharge instructional modality preference were assessed. Results Nearly one-quarter of the 2521 participants demonstrated low health literacy. Low health literacy predicted decreased 72-hour antibiotic retrieval (χ2 = 9.56, P =.008). No significant association with antibiotic retrieval was noted across the 3 treatment groups (χ2 = 5.112, P =.078). However, patients randomized to the text message group retrieved antibiotic prescriptions within 72 hours more frequently than did those randomized to the voicemail treatment group (χ2 = 4.345, P =.037), and patients with low health literacy randomized to voicemailed instructions retrieved their antibiotic prescriptions less frequently than did those randomized to standard of care instructions (χ2 = 5.526, P =.019). Reported instructional modality preferences were inconsistent with the primary findings of the study. Conclusions Discharge instructional modality impacts antibiotic retrieval in patients with low health literacy. Preference for discharge instructional modality varies by degree of health literacy, but does not predict which modality will optimize 72-hour antibiotic retrieval.