This study examined differences in students' classroom motivational climate perceptions and motivational beliefs between those enrolled in undergraduate Biology courses that implemented an innovative, active learning intervention and those enrolled in traditional Biology courses (control group). This study also sought to determine whether students' classroom motivational climate perceptions and motivational beliefs mediated the effect of the intervention on course grades. Participants were 962 college students attending a large US public university. Students self-selected into one of the biology courses were randomly assigned to the intervention or control group. Multiple regression analyses indicated that students receiving the intervention reported greater instructor support, a perception of higher expectations for understanding (academic press), and a greater feeling that the course was interesting (situational interest). They also held higher self-efficacy and value for their biology course at completion compared to their counterparts in traditional biology classrooms. Moreover, mediation analyses indicated that the effect of the intervention on course grades was better explained through students' classroom motivational climate perceptions and motivational beliefs. Results gained from this intervention may be useful to other campuses interested in enhancing student motivation and success even when faced with large enrolments and minimal faculty and staff support.