A gloss refers to a short definition or explanation of the meaning of a word in a text that can assist learners in reading comprehension and vocabulary learning. This study aims to investigate how glosses in English academic texts affect the reading behaviour of ESL undergraduates while reading is taking place. In addition, it also examines the effects of gloss on the learning of the glossed target words. Twenty ESL first year undergraduates who were grouped into proficient and less proficient learners based on their MUET (Malaysian University English Test) results participated in a reading experiment using the eye tracking device. Eye movement data (i.e. fixation duration and scan path) obtained from the eye tracker, retrospective interviews and three vocabulary tests were analyzed. The glossed target words were selected from Coxhead's Academic Word List (AWL) and were placed in the same line with the target word in the texts, in right margin of the texts. Eye movement analyses showed that the position of the gloss either at the top, middle, or bottom of the page influences how and when participants read the gloss. Participants who read the gloss before or after reading the texts were high unlikely to regress to the text to infer to the target words in the text. However, participants who read the gloss while reading the text mostly regressed to the text and read the target words in the text. The fixation duration data revealed that proficient participants looked at more glosses than the less proficient participants. The retrospective interview confirmed the fact that the participants did not read the gloss in each encounter as they have become familiar with the target words in the gloss. Nonetheless, they agreed that the presence of glosses in the texts helped them to learn unknown words, particularly in noticing the form of the words. The finding suggests that the use glosses need to be emphasized in language teaching and learning, particularly in reading comprehension and vocabulary learning. [For the full proceedings, see ED654100.]