Instructors create classroom environments that have the potential to impact learning by affecting student motivation, resistance, and self-efficacy. However, despite the critical importance of the learning environment in increasing conceptual understanding, little research has investigated what instructors say and do to create learning environments in college biology classrooms. We systematically investigated the language used by instructors that does not directly relate to course content and defined the construct of Instructor Talk. Transcripts were generated from a semester-long, cotaught introductory biology course (n = 270 students). Transcripts were analyzed using a grounded theory approach to identify emergent categories of Instructor Talk. The five emergent categories from analysis of more than 600 quotes were, in order of prevalence, 1) Building the Instructor/Student Relationship, 2) Establishing Classroom Culture, 3) Explaining Pedagogical Choices, 4) Sharing Personal Experiences, and 5) Unmasking Science. Instances of Instructor Talk were present in every class session analyzed and ranged from six to 68 quotes per session. The Instructor Talk framework is a novel research variable that could yield insights into instructor effectiveness, origins of student resistance, and methods for overcoming stereotype threat. Additionally, it holds promise in professional development settings to assist instructors in reflecting on the learning environments they create.