Purpose: This paper aims to examine the mediating role of employees' intrinsic motivation in the relationship between perceived ambidextrous organizational culture and innovative behaviour among information technology (IT) sector employees. Design/methodology/approach: The study used a quantitative research methodology, using a questionnaire to gather data from a sample of 510 employees across a range of IT organizations and various roles in the Indian IT sector. Smart partial least squares structural equation modeling Version 3 was used for the analysis and interpretation of the study. Findings: The findings revealed a significant positive relationship between perceived ambidextrous organizational culture and employees' innovative behaviour in the Indian IT sector. Moreover, the study established that employees' intrinsic motivation played a significant mediating role in this relationship. Originality/value: This study stands out for its exploration into how employees' intrinsic motivation mediates the relationship between ambidextrous organizational culture and innovative behaviour. It offers valuable insights for enhancing organizational creativity by understanding the critical role of intrinsic motivation.