Mixotrophy, a physiological trait combining autotrophy and heterotrophy in one organism, significantly contributes to energy and matter transfer in aquatic ecosystems. However, understanding how environmental factors influence mixoplankton success across freshwater ecosystems has been uncertain. The grand écart hypothesis (GEH) posits that light and nutrient availability are key components of mixotrophs' niche, suggesting that ecosystem properties determine opposing gradients of light and nutrients, creating environmental filtering for mixotrophs. We hypothesized that ecosystem size, a property of lake ecosystems, mediates the prevalence of patterns and control mechanisms predicted by the GEH on mixoplankton relative biomass (MRB). Using data from 98 tropical lakes, we demonstrated that lake size mediates the inverse relationship between light and nutrient availability across ecosystems. Larger lakes have more light but low nutrients, while smaller lakes have more nutrients but greater shading. Light availability better explains MRB in small lakes, and nutrients better explain MRB in large lakes, with MRB values being higher in small lakes, with secondary influence from zooplankton herbivory. Our results validate the GEH as a significant framework for explaining patterns and control mechanisms of mixoplankton across tropical lakes. This study highlights the significance of lake size as an ecosystem property that generates opposing light and nutrient gradients, further emphasizing its importance for understanding mechanisms regulating freshwater phytoplankton community structure and functioning. Integrating lake size within the conceptual framework of the GEH could aid in explaining mixoplankton success over macroecological scales.