Solanum nigrum fruit is like a treasure house for anticancer drugs because of its steroidal alkaloids. However, the clinical treatment of cancer mainly uses immature fruits, which can cause a toxic reaction if eaten directly, while mature fruits are eaten as fruit. In order to clarify the reasons for the differences in pharmacodynamics and toxicity between them, we studied the composition and metabolism of steroidal alkaloids in fruits of different maturities based on liquid chromatography‐tandem mass spectrometry and molecular networking. As a result, 114 steroidal glycoalkaloids were identified. During fruit ripening, the aglycones of steroidal alkaloids mainly undergo hydroxylation and carboxylation, and the sugar side chains mainly undergo acylation and glycosylation reactions. Furthermore, 219 steroidal alkaloids were identified in a metabolism experiment in rats. Metabolic processes include deglycosylation, redox, sulfuric acid binding, acetyl binding, and glucuronic acid‐binding. Steroidal alkaloids in mature fruits have high molecular weight and polarity, which are difficult to absorb, and most of them are excreted through feces and urine, which may be the reason for their poor efficacy. This study lays a foundation for research on the biosynthesis of steroidal alkaloids and provides potential candidates for the discovery of new steroidal alkaloid anticancer drugs.