Background Laminin, one of the largest glycoproteins of the basement membrane, is an important component of the extracellular matrix. Functions of the basement membrane include regulation of cell signaling behaviors and structural support. Laminin plays a critical role in the regulation of insulin action in muscle, liver, and adipose tissue. The study mainly investigates an association between the change in serum laminin levels and insulin resistance and non-alcoholic hepatosteatosis. Methods This prospective study included a total of 90 participants; 60 patients diagnosed with Grade 2-3 non-alcoholic hepatosteatosis and 30 age- and sex-matched healthy controls between December 2019 and December 2020. Routine laboratory tests including glucose, insulin, homeostatic model of assessment-insulin resistance (HOMA-IR), alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglyceride, low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and C-reactive protein and laminin levels were measured in the serum of the patient and control groups. Enzyme-linked immunosorbent assay was used for the measurement of laminin levels. Results The median serum laminin levels were lower in patients with hepatic steatosis, compared to the control group (72 ng/L vs. 82 ng/L, respectively; p = 0.003). In the patients with insulin resistance, median laminin levels were lower, regardless of the presence of non-alcoholic hepatosteatosis (67 ng/L vs. 85 ng/L, respectively; p = 0.007). There was a weak, negative correlation between the laminin levels and HOMA-IR. Conclusions Our study results suggest that, although there is no exact link between laminin and non-alcoholic hepatosteatosis, serum laminin levels are lower in patients with insulin resistance by regulating the insulin effect through integrins.