In pancreatic hypervascular masses, it is often difficult to differentiate pancreatic neuroendocrine neoplasm (PanNEN) and intrapancreatic accessory spleen (IPAS) before surgery because of their similar features, such as a round shape and early enhancement. This study aimed to examine the efficacy of endoscopic ultrasonography (EUS) for differentiating between them. This retrospective pilot study enrolled 136 patients with pathologically confirmed non-functional PanNEN or IPAS who underwent EUS at our institution. The clinical features, conventional EUS findings, EUS elastography (EUS-EG) findings, and contrast-enhanced harmonic EUS (CH-EUS) findings were retrospectively evaluated. In conventional EUS, calcification had significant differences between PanNEN and IPAS (P = 0.006). On EUS-EG findings (stiff/soft), patients with PanNEN had softer lesions (6 (25%)/18 (75%)) and those with IPAS had stiffer lesions (3 (100%)/0 (0%)) (P = 0.029). CH-EUS revealed that 4/4 (100%) patients with IPAS had hyperechoic or isoechoic vascular patterns up to 300 s, while only 1/15 (7%) patient with PanNEN had such patterns at 300 s (P = 0.001), resulting in significant washout after 180 s in PanNEN group. This study is the first report on EUS to differentiate between PanNEN and IPAS. It is useful to evaluate calcification with conventional EUS, stiffness with EUS-EG, and enhancement patterns with CH-EUS for that.