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The purpose of this exploratory study is to investigate students’ use and perceptions of online videos and intrinsic motivation toward the online videos in an organic chemistry laboratory course, which used online videos to assist students’ preparation and learning in the lab. Students’ responses to an anonymous survey showed that they watched the online videos one or more times based on their available time. Descriptive statistics showed that students had positive perceptions about the effect of online videos in terms of their preparation of labs, understanding the concepts, doing hands-on activities, and other aspects in the lab, which were consistent with their written comments. 44% of students showed positive intrinsic motivation toward the online videos according to items based on self-determination theory. Correlation results showed that the effect subscales were strongly related to each other, and students’ intrinsic motivation was significantly and positively related to students’ perceived effect of the online videos on their understanding and preparation with medium to large effect sizes. Inferential tests showed that students who had a positive perception of intrinsic motivation scored significantly higher on the understanding subscale with a medium to large effect size. The findings in this study suggest the online organic chemistry laboratory videos have significant potential to improve students learning and it is important to stimulate student intrinsic motivation toward the online videos which will help students benefits more from the effects of the online videos.