We evaluated the effects of altering the dietary ratio of C16:0 and cis-9 C18:1 on the fatty acid (FA) profile of plasma lipid fractions and adipose tissue (AT), and the association of dietary FA ratio with body weight (BW) change of lactating dairy cows. Cows (n = 24; 124 ± 30 DIM) were blocked by milk yield and assigned to FA treatments fed for 35 d. The FA treatments supplemented at 1.5% diet DM were (1) 80:10 (80% C16:0 + 10% cis-9 C18:1); (2) 73:17 (73% C16:0 + 17% cis-9 C18:1); (3) 66:24 (66% C16:0 + 24% cis-9 C18:1); and (4) 60:30 (60% C16:0 + 30% cis-9 C18:1). Subcutaneous AT samples from the tail region and blood samples from tail vein were collected on the last d of the study. The FA profile of AT and plasma phospholipids (PL), cholesterol esters (CE), triglycerides (TG), and free FA (FFA) determined. The statistical model included the random effect of cow and the fixed effect of treatment. Increasing cis-9 C18:1 in FA treatments increased the concentration of C16:0 (linear, P = 0.10), C18:0 (linear, P = 0.01), cis-9 C18:1 (linear, P = 0.01), trans-10 C18:1 (linear, P = 0.01), and cis-9, trans-11 C18:2 (linear, P = 0.01) in plasma TG. Increasing cis-9 C18:1 in FA treatments reduced C18:0 (linear, P = 0.01), and increased cis-9 C18:1 (cubic, P = 0.04) in plasma FFA. There were no effects of treatments on C16:0 and C18:0 concentrations in PL and CE (P > 0.25); however, increasing cis-9 C18:1 in FA treatments increased cis-9 C18:1 concentration in plasma PL and CE (both linear, P = 0.10). In AT, increasing cis-9 C18:1 in FA treatments increased the concentration of C18:0 (linear, P = 0.01) and cis-9 C18:1 (linear, P = 0.01), but reduced C16:0 (quadratic, P = 0.03). There were positive correlations between the concentration of cis-9 C18:1 in AT and BW change (r = 0.66, P < 0.01) and the concentration of cis-9 C18:1 in AT and plasma insulin (r = 0.44, P = 0.03). There were no correlations between BW change and AT concentration of C16:0 (r = −0.17, P = 0.44) or C18:0 (r = −0.04, P = 0.84). Our results demonstrate that altering the dietary ratio of C16:0 and cis-9 C18:1 changes the FA profile of AT and plasma lipid fractions. Changes in BW are positively associated with cis-9 C18:1 in AT and plasma insulin.