Conotoxin-Ac1 and its variant conotoxin-Ac1-O6P, were isolated from the venom duct of Conus achatinus, a fish-hunting cone snail species collected in the Sea of Hainan, China. Conotoxin-Ac1 is linear peptide that contain 15 amino acids. In the present study, we synthesized and structurally and functionally characterized conotoxin-Ac1 as well as 19 variants. Electrophysiological results showed that conotoxin-Ac1 inhibited N-methyl-D-aspartate receptor subunit 2B (NR2B) with an IC₅₀ of 8.22 ± 0.022 μM. Further structure-activity studies of conotoxin-Ac demonstrated that polar amino acid residues were important for modulating its active, and the replacement of N1, O9, E10, and S12 by Ala resulted in a significant decrease in potency to NR2B. °Furthermore, conotoxin-Ac1 and conotoxin-Ac1-O6P were tested in hot-plate and tail-flick assays to measure the potential analgesic activity to an acute thermal stimulus in a dose-dependent manner. Subsequently, the analgesic activity of conotoxin-Ac1 mutants was analyzed by the hot-plate method. The results show that N1, Y2, Y3, E10, N11, S12, and T15 play an important role in the analgesic activity of conotoxin-Ac1. N1 and S12 have significant effects on conotoxin-Ac1 in inhibiting NR2B and analgesic activity. In conclusion, we have discovered that conotoxin-Ac1 is an inhibitor of NMDAR and displays antinociceptive activity.