Neuromuscular blocking agents (NMBAs) are widely used in surgery to achieve skeleton muscles relaxation under light anesthesia status. In this work, we synthesized a series of 3,16-bisquaternary ammonium steroidal NMBAs. Among them, three compounds exhibited higher in vitro activities than the commenced drug rocuronium. In addition, structure–activity relationship was unveiled. We found that the intact acetylcholine-like moiety in D-ring was not necessary for maintaining activity but both the acetyl group and the quaternary nitrogen were very essential.