Problem‐based learning (PBL) is beginning to change the pedagogical principles of science education at the university level. Previously published data from our group found that PBL positively affects exam performance of low achieving students when proper support systems are provided (formative assessment and scaffolding). This study determined how motivation and learning strategy use (subject matter intrinsic value, self‐efficacy, self‐regulation, test anxiety, and cognitive strategy use) was affected in students of different academic backgrounds in response to differential types of lecture and PBL instruction. Students (n=92) in an upper level physiology course were randomly placed in one of three learning groups: traditional lecture‐style instruction (LI), guided problem‐based instruction (GPBI), or open problem‐based instruction (OPBI). Prior to instruction, students completed a 44 question 7‐point Likert scale survey based on the Motivated Strategies for Learning Questionnaire to obtain baseline data on student's motivational and self‐regulated learning strategies. A similar survey, with additional questions to identify the impact of key instructional factors and student perceived difficulties, was completed at semester's end. Cumulative grade point average (GPA), prior to the beginning of the class, identified students as low‐ (GPA <3), average‐ (3 ≤GPA <3.6), or high‐achieving (GPA ≥3.6). Students in the low‐achieving group significantly increased subject matter intrinsic value in GPBI and OPBI groups (p<0.05, Wilcoxon Rank sum). Low‐achieving students in the GPBI group had a significant decrease in test anxiety while low‐achieving students in the more intensive OPBI group significantly increased test anxiety (p<0.05). High‐achieving students showed significant increases in self‐efficacy, self‐regulation, and cognitive strategy use in both the GPBI and OPBI groups but a significant increase in test anxiety in the LI group (p<0.05). Average‐achieving students had significant increases in intrinsic value, self‐efficacy, self‐regulation, and cognitive strategies only in the LI group (p<0.05). When asked to rank the top five helpful instructional factors, students selected “Active learning” (LI: 43.8%, GPBI: 37%, OPBI: 52%), “Teacher enthusiasm” (LI: 41.9%, GPBI: 67.9%, OPBI: 48%), “In‐class discussion” (LI: 40.6%, GPBI: 53.6%, OPBI: 39.1%), and “Engaging students” (LI: 34.4%, GPBI: 36%, OPBI: 41.7%). LI students ranked “Focus on understanding” (28.1%) as the final factor, while GPBI students included “Sufficient explanation” (38.5%) and OPBI students selected “Interactive with students” (43.5%). Student identified learning difficulties included “Prefer slides with detailed notes” (LI: 9%, GPBI: 6%, OPBI: 8%), “Feel obligated to prepare for class” (LI: 0%, GPBI: 10%, OPBI: 8%), and “Fearful to make mistakes in class” (LI: 9%, GPBI: 0%, OPBI: 4%). Students identified similar instructional factors as helpful and had similar learning difficulties regardless of their individual learning group. OPBI and GPBI methods increase motivation and learning strategy use in both low‐ and high‐ achieving student groups. Low‐achieving students may benefit from a GPBI teaching methodology as test anxiety was decreased for this group. Average‐achieving students increased in motivation and learning strategy use only in the LI group, perhaps due to the familiarity of the traditional didactic learning environment.