Solanum nigrum fruits have been conventionally available as a material of beverage due to its nutritional substances such as minerals, vitamins, amino acids, proteins, sugars, polyphenols and anthocyanins. Here has rarely reported on the characterizaton of the components and the regulatory mechanism of Anthocyanins in S. nigrum. In this study, we determined that the peel and flesh of S. nigrum fruits shared the similar HPLC profiles, but the different contents and total antioxidant activities for Anthocyanins. After an efficient purification method mainly including extraction with pH 1.0 distilled water and then desorption with pH 1.0 95% ethanol after a DM-130 resin adsorption step to obtain more pure anthocyanins extracts, the purity of anthocyanins extract from S. nigrum fruits reached to 56.1%. Moreover, eight anthocyanins from S. nigrum fruit were identified with HPLC-MS/MS for the first time. A typical R2R3-MYB transcription factor gene, SnMYB, was also cloned for the first time by RACE-PCR from S. nigrum. Moreover, the contents of anthocyanins was shown a good correlation (r = 0.93) with the expression levels of SnMYB during the fruits developmental stages. Most significantly, SnMYB successfully produced high anthocyanins contents (1.03 mg/g) when SnMYB was transiently expressed in tobacco leaves. Taken together, S. nigrum fruits are a promising resource for anthocyanins extraction and SnMYB is an activator that positively regulates anthocyanins biosynthesis in S. nigrum.