(1) Background: To explore the anti-obesity effects and mechanisms of sika deer velvet antler peptides (sVAP) on 3T3-L1 preadipocytes and in high-fat diet (HFD)-induced obese mice. (2) Methods: sVAP fractions of different molecular weights were obtained via enzymatic hydrolysis and ultrafiltration. Their anti-lipid effects on 3T3-L1 cells were assessed with Oil Red O staining. The optimal fraction was tested in HFD-induced obese C57BL/6 mice to explore anti-obesity mechanisms. Peptide purification used LC-MS/MS, followed by sequence analysis and molecular docking for activity prediction. (3) Results: The peptide with the best anti-obesity activity was identified as sVAP-3K (≤3 kDa). sVAP-3K reduced lipid content and proliferation in 3T3-L1 cells, improved lipid profiles and ameliorated adipocyte degeneration in HFD mice, promoted the growth of beneficial gut microbiota, and maintained lipid metabolism. Additionally, sVAP-3K activated the AMP-activated protein kinase (AMPK) signaling pathway, regulating adipogenic transcription factors. sVAP-3K exhibited ten major components (peak area ≥ 1.03 × 10