Preimplantation factor (PIF) is a novel embryo-secreted immunomodulatory peptide. Its synthetic analog (sPIF) modulates maternal immunity without suppression. There is an urgent need to develop agents that could prevent the development of type 1 diabetes mellitus (TIDM). Herein, we examine sPIF's preventive effect on TIDM development by using acute adoptive-transfer (ATDM) and spontaneously developing (SDM) in non-obese diabetic (NOD) murine models. Diabetes was evaluated by urinary and plasma glucose, intraperitoneal glucose tolerance test (IPGTT), pancreatic islets insulin staining by immunohistochemistry and by pancreatic proteome evaluation using mass spectrometry, followed by signal pathway analysis. Continuous administration of sPIF for 4-weeks prevents diabetes development in ATDM model in >90% of recipients demonstrated by normal IPGTT, preserved islets architecture, number, and insulin staining. (P < 0.01). sPIF effect was specific; its protective effects are not replicated by scrambled PIF (χ(2) = 0.009) control. sPIF led also to increased circulating Th2 and Th1 cytokines. In SDM model, 4-week continuous sPIF administration prevented onset of diabetes for 21 weeks post-therapy (P < 0.01). Low-dose sPIF administration for 16 weeks prevented diabetes development up to 14 weeks post-therapy, evidenced by preserved islets architecture and insulin staining. In SDM model, pancreatic proteome pathway analysis demonstrated that sPIF regulates protein traffic, prevents protein misfolding and aggregation, and reduces oxidative stress and islets apoptosis, leading to preserved insulin staining. sPIF further increased insulin receptor expression and reduced actin and tubulin proteins, thereby blocking neutrophil invasion and inflammation. Exocrine pancreatic function was also preserved. sPIF administration results in marked prevention of spontaneous and induced adoptive-transfer diabetes suggesting its potential effectiveness in treating early-stage TIDM.