Glutathione S-transferases (GST) are a large family of polymorphous proteins that play important roles in herbicide detoxification and stress response. Nicosulfuron is the most broadly used herbicide in maize fields, and it can cause different injuries to maize varieties, but little is studied about the systemic and comprehensive GST gene family responding to nicosulfuron stress in maize. In this research, pre-treatment with glutathione S-transferase inhibitor 4-chloro-7-nitrobenzoxadiazole (NBD-Cl) increased nicosulfuron phytotoxicity to both sensitive and tolerant maize genotypes. A total of 55 ZmGST genes belonging to six major sub-classes were identified in the maize genome and named according to the nomenclature system. Based on phylogenetic analyses, highly conserved gene structure and motif distribution were detected in the same class. Chromosome mapping results showed that ZmGST genes were distributed over the 10 chromosomes unevenly. There were thirteen and eight gene pairs identified as tandem and segmental duplication events, respectively, which played important roles in the expansion of the GST gene family in maize. RNA-seq and qRT-PCR analyses showed that there were great dissimilarities in ZmGST gene expression patterns between the tolerant and sensitive maize plants. More highly expressed ZmGST genes were found in the tolerant than in the sensitive without nicosulfuron stress. However, under 60 g a.i. ha−1 nicosulfuron stress, more ZmGST genes were significantly upregulated in HB41 than in HB09. This study provided experimental evidence showing that glutathione S-transferases were involved in nicosulfuron stress in maize. It will contribute to the further functional analysis of the GST gene family in maize.