Previous research has reported the importance of students' perceptions of learning environment in their affective outcomes, motivational regulations, and performance. In this study, we investigated the predictive effects of students' perceptions of biology classroom learning environment on their mastery goals orientation, motivational regulations, and performance in biology. A total of 329 Iranian high school students completed questionnaires on perceptions of biology classroom learning environment, personal mastery goal orientation, and, intrinsic and extrinsic regulations in learning biology. The results revealed that perceived meaningfulness predicted personal mastery goals orientation moderately and positively. Perceived autonomy had significant and positive influences on intrinsic and extrinsic regulations. The results also showed that students' perceived meaningfulness and intrinsic regulation were slightly and positively correlated. Personal mastery goals orientation was moderately and positively correlated with students' intrinsic and extrinsic regulations. Moreover, the results indicated that perceived competition, perceived meaningfulness, personal mastery goals orientation, and extrinsic regulation positively affected students' performance in biology. The findings also underscore the central mediating role of students' personal mastery goals orientation in the relationship between perceptions of learning environment and their regulations in biology learning. The results support many of the hypothesized relationships and provide broader clarification and implications for the literature.