Background: Percutaneous ultrasound guided trucut needle biopsy (US-TNB) is an important method for diagnosing pancreatic space-occupying lesions. However, it is uncertainty about which needle size is more suitable. There are few reports in the past. We aimed to evaluate the performance of the 18- and 20-gauge (18G and 20G) types of needle in obtaining histopathological diagnosis of pancreatic space-occupying lesions. Methods: This study included 285 patients (70: 18G needle/215: 20G needle) who underwent ultrasound-guided real-time puncture of pancreatic space-occupying lesions in the Ultrasonic Center & Xijing Hospital of Digestive Diseases, the Air Force Medical University from January 2009 to June 2017. Two types of needle were compared with respect to technical success rate, diagnostic rate, accuracy, sensitivity (Se), specificity (Sp), positive predictive value (PPV) and negative predictive value (NPV) for histolopathological diagnosis. Results: Percutaneous US-TNB histopathological findings were compared with the gold standard of surgical histopathological findings or long-term clinical follow-up. The puncture technical success rate was 100% for both 18G and 20G needle. The diagnostic rate of 18G and 20G were 91.4% and 84.7% respectively, with no significant difference between them (P>0.05). The accuracy of 18G needle was 96.8% and that was 96.7% for 20G needle, the difference was not statistically significant (P>0.05). The Se, Sp, PPV, and NPV were 96.3%, 100%, 81.3%, and 18.8% for the 18G needle compared with 96.0%, 100%, 76.9%, and 23.1% respectively for the 20G needle. No serious complications occurred. Conclusion: Current data show no significant difference between the 18G and 20G needle in diagnostic rate and accuracy for the diagnosis of pancreatic space-occupying lesions. However, the 18G needle establishes the diagnosis with fewer puncture times.