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With the rise of online instruction, a better understanding of the factors that contribute to belonging and motivation in these contexts is essential to creating optimal learning environments. Although group work is known to be beneficial to student success, few studies have investigated its role in the context of asynchronous online courses. The present study addresses this gap through a survey of 146 undergraduate students in an asynchronous online physiology lab over two semesters, one with required group work and one without group work. Students were surveyed to evaluate the influence of group work on their motivation and sense of belonging, as well as their perceptions of inclusive and exclusive features of the course. Students assigned to groups had a higher sense of belonging (P = 0.006) and beliefs about their competence (P = 0.002) and perceived lower effort and psychological costs associated with the course (P = 0.04 and 0.04, respectively) compared to students not assigned to groups. Students assigned to groups reported that peer interactions made them feel included in the course (70% of coded responses) while those not assigned to groups valued instructor interactions (51% of coded responses) as inclusive. Negative peer interactions were commonly reported as exclusive by students assigned to groups (28% of coded responses) while a lack of peer interactions (23% of coded responses) made students not assigned to groups feel excluded. These data indicate that assigning groups in asynchronous online courses is an effective way to increase student motivation and perceptions of belonging.