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Background: Pancreatic acinar cell carcinoma (PACC) is a rare type of pancreatic exocrine neoplasm that is frequently diagnosed at late stages with a high rate of metastasis. Identification of new biomarkers for PACC can improve our knowledge of its biology, early detection, or targeted therapy. In this study, hybridoma technology was used to generate mAbs against Faraz-ICR, a pancreatic acinar cell carcinoma cell line., Methods: Cell ELISA and flow cytometry were used for screening, and the 4H12 hybridoma clone was selected for further analysis. The 4H12 mAb was specific for myosin heavy chain-9 (MYH9) as determined by Immunoprecipitation, Western blot, and mass spectrometry., Results: This antibody reacted variably with other cancer cells, in comparison to Faraz-ICR cell. Besides, by immunohistochemical staining, the acinar cell tumor, which was the source of Faraz-ICR, showed high MYH9 expression. Among 21 pancreatic ductal adenocarcinoma cases, nine (42.8%) expressed MYH9 with low intensity, while 10 (47.8%) and 2 (9.5%) cases expressed MYH9 with moderate to strong intensities, respectively. The 4H12 mAb inhibited the proliferation of Faraz-ICR cells in a dose-dependent manner from 0.75 to 12.5 μg/ml concentrations (p < 0.0001 and p < 0.002). IC50 values were achieved at 12.09 ± 4.19 µg/ml and 7.74 ± 4.28 µg/ml after 24- and 48-h treatment, respectively., Conclusion: Our data suggest that the 4H12 mAb can serve as a tool for investigating the role of MYH9 pancreatic cancer biology and prognosis.