This paper examines the extent to which gender influences on the practice of Entrepreneurial Orien-tation (EO) on Start-up Success. This study was conducted among Spin-off and Symbiosis Com-pany (SSC) from all sectors in Peninsular Malaysia. Despite the remarkable research on EO and Start-up firms, there is a missing link on how gender provides different perspectives of EO practic-es among SSC, especially in Malaysia. A total of 120 SSC was chosen and the results show that EO was statistically related to Start-up Success and unveiled the magnitude of change that gender possesses in improving the relationship between EO practice and Start-up Success. The major implication of this study presents the unique contribution of gender in motivating SSC owners to engage in EO. Also, the difference of gender perceptions in business provides a variety of untapped opportunities in terms of entrepreneurial-related practices as different gender perceived different needs and capabilities. Future research is suggested to explore this phenomenon more extensively and develop a comprehensive model for the gender analysis. In brief, the discussion in this paper would help to strengthen the body of knowledge on Entrepreneurship and act as a future reference on SSC, EO, and Start-up Success.