Enolase (ENO), as an enzyme in the glycolytic pathway, is involved in plant development and can respond to abiotic stresses. In this study, the cadmium (Cd) hyperaccumulator plantpokeweed (Phytolacca americana L.) was used as material. According to the transcriptome data of pokeweed, the specific primers were designed and the PaENO gene was isolated from pokeweed leaves. Then the sequence analysis, prokaryotic expression and purification, expression pattern analysis and Cd resistance assay were performed. This will provide a basis for further study on the enzymatic activity of PaENO protein and the mechanism of PaENO gene in response to Cd stress. The results showed that: (1) PaENO gene (GenBank accession number JN656932.1) was successfully cloned and the open reading frame (ORF) of the gene was 1 335 bp, which encoded 444 amino acids. (2) The multiple sequences alignment analysis indicated the identity of PaENO protein and McENO protein of Mesembryanthemum crystallinum was the highest, which reached 93.47%. Phylogenetic analysis showed that PaENO protein had closest homology with M. crystallinum, Beta vulgaris and Spinacia oleracea, and they belonged to the same evolutionary branch. (3) Realtime PCR results showed that the expression level of PaENO gene was the highest in pokeweed leaves, 2.5 times that of roots, followed by that in roots, and the lowest in stems, only 1/5 of that in roots. After Cd treatment, the expression level of PaENO gene was significantly increased. At 2 h, the expression level of PaENO gene rapidly increased to 9.54 times of that before Cd treatment (0 h), and decreased to 3.12 times and 2.89 times of that (0 h) after Cd treatment at 12 h and 24 h, respectively. This indicated that PaENO gene was involved in the response to Cd stress in pokeweed. (4) The prokaryotic expression vector pET28aPaENO was successfully constructed and transformed into E. coli BL21(DE3) competent cells. After induction by IPTG, the target protein band appeared at about 50 kD. Then, the E. coli cells were disrupted by sonication, and the supernatant and precipitate were collected after centrifugation. SDSPAGE analysis showed that PaENO was expressed in both the supernatant and the precipitate, and the expression level of PaENO was higher in the supernatant. The purified PaENO protein was obtained by using Ni affinity chromatography kit. The results of Cd resistance assay preliminarily confirmed that the expression of PaENO protein can significantly improve the resistance of E. coli to Cd. The study suggested that PaENO protein may play a regulatory role in pokeweed to cope with Cd stress in the form of transcription factor (PaMBP1).