Mobile in-app advertising has become one of the most popular business advertising platforms in recent years. This relatively new advertising strategy’s annual spending has increased dramatically in recent years. Despite its practical success, the background theory for mobile in-app advertising is still in its infancy. This type of advertising also sees the emergence of new participants, such as ad networks and app publishers, which leads to the development of new theoretical constructs and more nuanced conceptual relationships on top of popularly accepted frameworks, e.g. Mobile Advertising Effectiveness Framework. This study especially looks at app publishers’ roles, which have received the least attention in the advertising literature and are almost non-existent in the previous effectiveness frameworks. The empirical goal of this study was to assess the moderating effects of ad space characteristics on the effectiveness of mobile in-app advertising. To test the research’s conceptual model, a 24-factorial online experiment was set up. Accordingly, this study established a common goal for participants and a metric to measure that common effectiveness goal. Ultimately, the model was successfully tested by both Moderated Regression Analysis and Multigroup Moderation Analysis techniques with data from over 15,000 ad impressions and over 800 ad clicks from thousands of mobile users in over 160 countries around the world. The study theoretically found that publishers can significantly moderate the effects controlled by advertisers, and users, which can practically help increase the revenue of advertising through designing and delivering ad spaces on their applications.