Computational Thinking (CT) has evolved as an essential competency for K-12 students, and programming practices are recognized as the key way to facilitate CT development. However, most studies of CT development in middle graders have focused on visual programming, lacking evidence to demonstrate the effectiveness of Python programming. Therefore, this study first developed 16 Python programming courses based on the CodeCombat programming platform. Then we conducted a 16-week Python programming course intervention study with 79 middle school students in seventh, eighth, and ninth grades in China to clarify the impact of Python programming on middle graders' CT. The results revealed that Python programming intervention significantly improved CT for seventh, eighth, and ninth graders. The analysis of interaction effects for grade and gender showed that Python programming was most beneficial for eighth graders in CT improvement. Meanwhile, we found that the difference in CT caused by the gender factor varied according to grade level. Specifically, the gender factor caused significant CT differences in eighth and ninth graders, and girls were better performers than boys. These findings enrich the outcomes of Python programming and CT education in middle schools and provide implications for frontline programming educators to conduct Python interventions.