Carotenoids in tomatoes confer significant health benefits to humans but with the disadvantage of the carotenoids from raw tomatoes not being easily absorbed for utilization. Thus, this study aimed to investigate the effects of different cooking processes on carotenoid release and human gut microbiota composition during in vitro simulated gastrointestinal digestion of tomatoes. The results showed that stir-frying significantly increased the release of lycopene and β-carotene during gastrointestinal digestion, with boiling being the second most effective treatment. The boiling-treated tomatoes enhanced the carotenoid release during in vitro fermentation. Gut microbiota analysis revealed that the digestion of the raw and boiled tomatoes promoted the growth of potentially beneficial microbiota while reducing the ratio of Firmicutes/Bacteroides, which potentially helps prevent obesity. Boiling treatment significantly reduced the growth of Peptostreptococcus and was negatively correlated with carotenoid release. Overall, the boiling-treated tomatoes were more effective than the raw or stir-fried tomatoes in terms of both colon health benefits and carotenoid release.