Serine protease inhibitor Kazal type 1 (SPINK1) was originally identified as a trypsin inhibitor in the pancreatic acinar cells in 1948. Recent studies showed an association of mutations in SPINK1 gene and hereditary chronic pancreatitis. Thus, a lack of SPINK1 may result in the premature conversion of trypsinogen into active trypsin in acinar cells, leading to pancreatitis. However, we found that mice deficient for Spink3, a mouse homologue of SPINK1, died after birth due to excessive autophagy (cellular self-digestion) in the pancreatic acinar cells, suggesting that Spink3 is involved in the regulation of autophagy. We further demonstrated that autophagy is involved in trypsinogen activation within the pancreatic acinar cells in experimentally induced pancreatitis. These results suggest that Spink3 has protective roles in pancreatitis by dual mechanisms, one as a trypsin inhibitor and a second as a suppressor of trypsinogen activation through negative regulation of autophagy. On the other hand, SPINK1 is structurally similar to epidermal growth factor (EGF), in terms of the number of amino acid residues and the presence of 3 intrachain disulfide bridges. In fact, Spink3 acts as a growth factor in various cell lines in vitro. To gain additional insight into the new function of Spink3 in vivo, we examined the expression pattern of Spink3 during development. We found that Spink3 was expressed in unexpected tissues such brain and mesonephric tubules. In this review, we summarize the old and new roles of SPINK1/Spink3 in trypsin inhibition, autophagy, and cell proliferation/differentiation.