Natural α-glucosidase inhibitors (aGIs) are of great interest as an efficacious and safe therapy for type 2 diabetes, which is an ongoing global health issue. The aim of this study is to utilize shrimp head powder (SHP), an abundant and low-cost material, for the biosynthesis, isolation, and identification of active antidiabetic compounds. SHP was efficiently converted to aGIs via Paenibacillus sp. TKU042 fermentation. Fermented SHP (fSHP)
by this strain possesses high pH stability, and stronger yeast α-glucosidase inhibitory activity (92%) than that of acarbose (60%). aGI productivity increased more than 2-fold after optimization (from 225 U/mL to 560 U/mL). Further bioactivity-guided isolation of two major active compounds were identified as nicotinic acid and adenine. Notably, these inhibitors were non-sugar-based moiety aGIs, which is newly isolated and identified from fSHP in this study. In the tests of specific enzyme inhibitory activity, adenine showed highly specific inhibition against yeast α-glucosidase (IC50 = 22 μg/mL); nicotinic acid demonstrated good effect on rat α-glucosidase (IC50 = 70 μg/mL); while acarbose possessed efficient effect on bacteria, rice, and rat α-glucosidases (IC50 = 0.03–108 μg/mL). The current results suggest that it is cost-effective to produce potent aGIs from SHP via Paenibacillus conversion, and these active constituents may be useful in type 2 diabetes management.