Bovine milk, a rich source of proteins and fatty acids, has a beneficial impact on physicochemical and organoleptic properties. The current study was planned to identify oxytocin's effects on protein and fatty acid profiles of cow milk (the Sahiwal breed) at various lactation stages (early, middle, and late) under a controlled atmosphere and feeding inputs. Examination of milk protein and fatty acid profiles by urea polyacrylamide gel electrophoresis and gas chromatography-mass spectrometry respectively revealed the significant effect (P < 0.01) of oxytocin during the cow's lactation stages. In electrophoretic patterns, casein (αs1-CN, αs2-CN, β-CN, and k-CN) and whey protein fractions (Ig, BSA, β-Lg, and α-La) appeared as low-intensity bands in oxytocin-injected milk when compared to the control. Reductions in concentrations of fatty acids C16:0 (25.00%) and C18:1 (21.60%) were observed in oxytocin-treated milk as compared to the control group (29.30% and 29.10%, respectively) at late lactation.