This study aimed to evaluate the functional and probiotic properties of Lactobacillus acidophilus SJLH001 (La-SJLH001) isolated from fermented food via mechanism analysis based on transcriptomics and culturomics. La-SJLH001 exhibited good tolerance to acid and bile salt conditions with anti-diabetic ability and cholesterol assimilation activity in vitro. Supplementation with La-SJLH001 also resulted in a significant reduction in host oral glucose and serum total cholesterol levels in vivo. Transcriptome profiles and qPCR analysis suggested that La-SJLH001 significantly regulated the transcription of key genes involved in glucose transportation, cholesterol metabolism, ion channels, and immune response, resulting in improved glucose homeostasis and cholesterol metabolism. La-SJLH001 significantly affected the structure of intestinal microbiota when analyzed by using culturomics coupled with MALDI-TOF MS. These results indicated that La-SJLH001 may be a mechanistic target for the control of diabetes with great potential in the application of probiotic products.