One of the macronutrient components of breastmilk is protein, which is very beneficial in infant growth and development. Maternal, infant and physiological factors affect protein levels in breast milk. One of the maternal factors is body mass index (BMI). This study was conducted with the aim to determine the effect of BMI on protein levels in expressed breast milk. The design of this study was observational analytic with a cross sectional approach. The research was conducted at the Medical Basic Chemistry Laboratory, Faculty of Medicine, Universitas Sriwijaya in May-November 2023. This study used 63 samples of expressed breast milk from breastfeeding mothers in Palembang city aged 20-35 years with babies aged 1-6 months who met the inclusion criteria. The 63 breast milk samples were divided into 2 categories, 30 breast milk samples from mothers with normal BMI and 33 breast milk samples from mothers with obese BMI. Protein was measured using the Lowry method and data were analyzed using the Mann-Whitney test. The average protein content of breast milk from mothers with normal BMI was 6.35±5.78 mg/mL and the average protein content of breast milk from mothers with obese BMI was 8.64±3.70 mg/mL. In the Mann-Whitney test results obtained p=0.03 (p0.05), thus there is an influence of maternal IMT on protein levels in breast milk. In conclusion, maternal BMI affects protein levels in breast milk.