Differential diagnosis between benign and potentially malignant cystic pancreatic lesions may be difficult. Previously we have compared cyst fluid serine protease inhibitor Kazal type I (SPINK1) with some traditionally used tumour markers (amylase, CEA, Ca19-9) and found that it may be a new promising maker in the differential diagnosis of cystic pancreatic lesions. In the present study, we focused on cyst fluid SPINK1 levels in benign and potentially malignant cystic pancreatic lesions.Sixty-one patients operated on for cystic pancreatic lesion in Tampere University Hospital, Finland and in Verona University Hospital, Italy, were included. Cyst fluid was aspirated during surgery, stored at −70 °C, and analysed with immunofluorometric assay for SPINK1. The final diagnosis was acute pancreatitis with fluid collection (Acute FC) in 4 patients, chronic pseudocyst (PS) in 17 patients, serous cystadenoma (SCA) in 7 patients, mucinous cystadenoma (MCA) in 21 patients and intraductal papillary-mucinous neoplasm (IPMN) in 12 patients (9 main/mixed duct type and 3 branch duct type).The acute FC patients had high SPINK1 levels. Among chronic cysts, SPINK1 levels were significantly higher in patients with potentially malignant cysts (main/mixed duct IPMN and MCA) than with benign cysts (side branch IPMN and SCA), (median and range, [480 (13–3602) vs. 18 (0.1–278) μg/L]; p < 0.0001). In the subcohort of 24 patients with <3 cm chronic cyst, cyst fluid SPINK 1 levels were significantly lower in SCA or side branch IPMN (3 [2–116] μg/L) than in main duct IPMN or MCA (638 [66–3602] μg/L; p = 0.018). The best sensitivity and specificity to differentiate any size MCA or main/mixed type IPMN from SCA or side branch IPMN were 85% and 84% (AUC 0.94; cut-off value 118 μg/L). The best sensitivity and specificity to differentiate <3 cm MCA or main duct IPMN from SCA or side branch IPMN were 93% and 89% (AUC 0.98; cut-off value 146 μg/L).Cyst fluid SPINK1 may be a possible marker in the differential diagnosis of benign and potentially malignant cystic pancreatic lesions.