MicroRNAs (miRNAs) play key roles in regulating zinc (Zn) toxicity tolerance in plants. Solanum nigrum is a typical Zn/Cd-accumulating plant that has a high Zn/Cd tolerance. Despite their importance, no miRNAs have been identified from S. nigrum thus far. In this study, small RNA sequencing was used to identify Zn-responsive miRNAs in S. nigrum roots. We identified 176 differentially expressed miRNAs in S. nigrum roots in response to Zn toxicity. We also found that all these differentially expressed Zn-miRNAs simultaneously respond to the exogenous NO donor sodium nitroprusside (SNP), indicating that NO is involved in Zn-mediated miRNA expression inS. nigrum. These differentially expressed miRNAs are involved in regulating the following processes in S. nigrum roots: phenylpropanoid catabolic and metabolic processes; lignin catabolic and metabolic processes; and programmed cell death in response to ROS. These results suggest that these miRNAs play key roles in the Zn toxicity tolerance of S. nigrum.