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Distinction of small-sized hepatocellular carcinoma (HCC) from dysplastic nodules may be difficult. In addition, distinction of well-differentiated HCC (WD-HCC) from high-grade dysplastic nodule (HGDN) is also difficult in small needle biopsy. We aimed to study serine peptidase inhibitor, Kazal type 1 (SPINK1) immunohistochemical expression in HCC to differentiate it from nonmalignant lesions.This study included 179 specimens from the archival material of Pathology Department, National Liver Institute, Menoufia University, between 2007 and 2014, divided as 93 HCC and 86 nonmalignant lesions. All cases were stained for SPINK1 antibody.SPINK1 was expressed in 76.3% of HCC cases with a diagnostic accuracy of 79.3%.There was a significant difference between focal nodular hyperplasia and WD-HCC cases regarding mean value of SPINK1 expression (P=0.015). In addition, there was low SPINK1 score in cirrhosis cases compared with WD-HCC. Moreover, there was a high significant difference between WD-HCC and HGDN regarding SPINK1 expression (P=0.001), with 83.3% sensitivity and 84.6% specificity.SPINK1 can be used to differentiate between a WD-HCC and a HGDN with high diagnostic validity.