QX-314 has been shown to produce long-acting local anesthesia in vivo in animals; however, translation to humans has been impeded by concerns about toxicity. We investigated whether the newly emerged QX-OH molecule could confer long-lasting anesthesia with a low local toxicity in rats. In rat sciatic nerve block model, QX-OH 25 mM produced a longer sensory block than QX-314 25 mM (median [25th, 75th percentiles], 5.5 [4.25, 6] h vs. 3 [3, 4] h; P = 0.03). QX-OH 35 mM produced a longer sensory block than QX-314 35 mM (8 [6, 12] h vs. 6 [4, 6.5] h, P = 0.038). QX-OH at 35 and 45 mM generated longer motor blocks than QX-314, with tissue toxicity less than that of QX-314 at the same concentration. In contrast with bupivacaine, QX-OH was clearly superior in terms of sensory and motor blockade durations after a single bolus injection. There was no significant difference in tissue toxicity between QX-OH (25 and 35 mM) and bupivacaine. In rat cutaneous trunci pinprick model, the QX-OH-induced pain threshold remained significantly different from baseline at 6 h (25 mM, P < 0.0001), 10 h (35 mM, P < 0.0001), and 12 h (45 mM, P < 0.0001). The time required for full recovery from the subcutaneous anesthetic effect was significantly longer for QX-OH than for QX-314 and bupivacaine. So QX-OH produced concentration-dependent, reversible, and long-acting local anesthesia in animal models with a moderate local toxicity.