Critical thinking is the way students think about the subject, content or problem that is able to improve their thinking skills skillfully. This study aims to determine the effect of the Virtual laboratory media on critical thinking in students on the subject of Biodiversity. This research was conducted at SMA Negeri 2 Babat Supat. The research method used is Pre-Experimental Design with One-Group Pretest-posttest design. The data collection technique used total sampling, with a sample of 49 students. The pretest-posttest instrument consisted of 20 multiple choice questions based on critical thinking indicators. The data analysis technique used the One Sample Kolmogorov Test with a data significance of < from = 0.05. The results of this study showed that the average pretest was 40.82, the posttest score was 85.31 and Ngain was 0.75 in the high category. From the results of hypothesis testing, the posttest value was 0.01 with a data significance of = 0.05. Then there is an influence on the Virtual laboratory learning media on the critical thinking of class X students, thus H0 is rejected and Ha is accepted. Keywords:Critical Thinking, Learning Media Virtual laboratory