Ultra-high performance liquid chromatography coupled with photo-diode array detector and electrospray ionization-mass spectrometry was employed to analyze the major fatty acids in Spirulina platensis 95% ethanol extract (SPL95). The effects of SPL95 on hepatoprotection were evaluated, including liver tissue histopathology, liver, and serum biochemical analysis. The active principle of SPL95 revealed a hypolipidemic effect, as indicated by down-regulating the mRNA and protein levels of sterol regulatory element-binding transcription factor-1c, 3-hydroxy-3-methyl glutaryl coenzyme A reductase, acetyl CoA carboxylase pathway, and upregulating adenosine 5′-monophosphate-activated protein kinase-α in liver. SPL95 enriched the beneficial bacteria, including Prevotella, Alloprevotella, Porphyromonadaceae, Barnesiella, and Paraprevotella. Treatment with SPL95 led to a decrease in microbes, such as Turicibacter, Romboutsia, Phascolarctobacterium, Olsenella, and Clostridium XVIII, which were positively correlated with serum triglyceride, total cholesterol, and low-density-lipoprotein cholesterol levels, but negatively correlated with the serum high-density-lipoprotein cholesterol levels. These results provide evidence that the fatty acid from SPL95 may be used as a novel adjuvant therapy and functional food to regulate gut microbiota in obese and diabetic individuals.