The study investigated the influence of ethnicity, gender and grade level on the motivational beliefs and self-regulation in Biology learning through a non-experimental quantitative study among 317 students in 2 co-educational schools in Nakuru (n = 155 ) and Siaya (n = 162) counties, Kenya. The study used a modified version of Motivated Strategies for Learning Questionnaire (MSLQ) which had 44 items to measure motivational beliefs and self-regulation. A 4 × 2 × 2 MANOVA revealed “ethnicity” as having the largest effect on these variables. There was a statistically significant difference between the Nakuru group and the Siaya group in favor of Nakuru with regard to self-efficacy in Biology learning. They also scored higher in most of the variables. There were no statistically significant gender differences in motivational beliefs and self-regulation in Biology learning. However boys were more self-efficacious with low test anxiety than girls. Girls had higher intrinsic value, cognitive strategy and self-regulation than boys. There were statistically significant grade level differences in favor of grade 12 students. Grade 11 students had the lowest scores in all the variables under investigation. The implications for the study are discussed.