Animal dissection has been routinely practiced in American biology classrooms for decades. With technological advancements, more states adopting student choice measures, and increased awareness about ethical concerns surrounding dissection, many useful dissection alternatives have been developed. To understand the current use of animal dissection and alternatives, and attitudes toward the practices, a nationwide survey of middle and high school biology teachers (n = 1178) and students (n = 500) was conducted. Most teachers (84%) and students (76%) reported using dissection in their classrooms, although nearly half of educators indicated that dissection is decreasing at their school. Educators cited student performance as the main factor driving their decision to use dissection or alternatives and reported conducting dissection exercises because of student interest. Most teachers had an interest in using alternatives, although only 36% used them in place of dissection. More than a third of biology students preferred the use of alternatives over animal specimens, yet most did not request dissection alternatives. Enabling students to opt in to dissection exercises rather than opt out, as is the current practice, and educating teachers and students about student choice and the advantages of dissection alternatives are suggested strategies to reduce animal use in education, in line with the “3 R’s” principle.