Innovativeness is one of the crucial factors allowing companies to grow, and innovative behavior of entrepreneurs is an important source of firm innovativeness and business success. This study aims to better understand self-regulatory mechanisms stimulating the innovative behavior of entrepreneurs. We have tested the mediation model in which work-related affect (enthusiasm, comfort, anxiety, and depression) mediates the relationship between work self-efficacy and innovative behavior. A longitudinal study with three measurement times was conducted on a sample of entrepreneurs-business owners. The mediation analysis with bias-corrected bootstrapping method confirmed that the relation between work self-efficacy and innovative behavior of entrepreneurs is mediated by their work-related positive affect--comfort and enthusiasm--but not by negative affect.