Objective To evaluate the efficacy and safety of the second session of endoscopic ultrasound-guided lauromacrogol ablation (EUS-LA) for pancreatic cystic neoplasms (PCNs). Methods A total of 74 patients with suspected of PCNs who underwent EUS-LA in the First Medical Center of Chinese PLA General Hospital from April 2015 to December 2020 were enrolled in the study. Fifteen of them underwent the second ablation. The efficiency of EUS-LA was determined based on the change of lesion volume during the imaging follow-up, categorized into complete resolution (CR), partial resolution (PR) and stable disease (SD). The safety was determined by the complication incidence. Results Among the 15 patients undergoing the second EUS-LA, there were 9 males and 6 females with age of 51.5±17.6 years. Thirteen patients completed imaging follow-up after the second EUS-LA, CR was achieved in 8 patients, PR in 2 patients and 3 in SD. The CR rate increased from 0 after the first ablation to 8/13 after the second ablation (P=0.002). The median cyst diameter decreased from 22.0 mm before the second ablation to 15.0 mm after the second ablation (Z=-2.666, P=0.008) and the median cyst volume reduced from 2 419.7 mm3 to 1 099.5 mm3 (Z=-2.134, P=0.033). The complication incidence of the second ablation was 2/15, similar to that of the first ablation. Conclusion The second ablation is effective and safe without increasing the complication incidence in patients without achieving CR after the first EUS-LA.