The study aimed at determining the effects of aspects of self-regulated learning (SRL) such as metacognition and motivation on reading comprehension. A nationwide, representative sample (N = 6,403) of 15-year-old Greek adolescents was drawn from the PISA 2018 database. The participants’ data on metacognitive knowledge of reading strategies, reading task-specific metacognitive experiences, intrinsic motivation, and reading comprehension were selected for subsequent analyses. Multiple-group confirmatory factor analyses were conducted to test gender differences in metacognitive functioning via measurement invariance. Structural equation modeling was also utilized to assess predictive and mediating effects between motivation, metacognition and reading comprehension achievement. Results indicated gender-related individual differences in metacognitive functioning. Further, structural equation modeling showed that metacognitive experiences, metacognitive knowledge and intrinsic task motivation predicted reading comprehension achievement; however, metacognition mediated the association of intrinsic motivation with reading comprehension achievement. Potential cognitive and educational implications are briefly discussed