Magnesium is absorbed in the rumen but is antagonized by high dietary K. Because the kidney regulates Mg, urinary Mg can be used as an index of Mg absorption. The rumen environment also affects Mg absorption and milk fatty acid (MFA) profile. Therefore, the objective of this experiment was to investigate the interaction of dietary K and Mg on transient Mg metabolism and milk fatty acid profile in dairy cows. Thirty-two multiparous cows (215 ± 32 DIM) were placed into 8 blocks based on milk yield (33.1 ± 8.8 kg/d). Cows were fed a common diet (0.24% Mg and 1.74% K on a DM basis) for 7 d and then abruptly switched to 1 of 4 treatments for 1 wk: basal Mg, basal K (Basal; 0.21% Mg and 1.73% K); basal Mg, High K (HiK; 2.71% K), High Mg, basal K (HiMg; 0.43% Mg) and High Mg, High K (HiK+Mg; 0.44% Mg and 2.81% K). Supplemented diets contained 2.0% K₂CO₃ and/or 0.36% and MgO. Spot urine samples were taken on d0, 2, 4 and 7 to measure Mg and creatinine and predict excretion of urinary Mg. Milk was collected on d7 for MFA profile. Data were analyzed with a model containing the fixed effects of K, Mg, K x Mg, and the random effect of block. For urine measures, the fixed effect of day (repeated) was added with d0 as a covariate. In milk fat, K decreased the proportion of C4 and C6 (P < 0.01) while Mg increased iso 16:0 (P < 0.05) and tended to increase trans-10,cis­-12 18:2 and total iso­-FA (P < 0.10). Anteiso 15:0 increased in HiK (5.61 mg/kg) and HiMg (5.58 mg/kg) but was not different between HiK+Mg (5.14 mg/kg) and basal (5.28 mg/kg). A K × Mg × day interaction on urinary Mg excretion (P < 0.05) was found. Excretion of Mg did not change over time for basal diet (8.7 g/d), but supplementing K without Mg decreased excretion on d 2 and 4 (5.2 g/d) and then increased on d 7 (6.6 g/d). Feeding supplemental Mg linearly increased Mg excretion over time. When both K and Mg were supplemented, Mg excretion increased on d 2 (11.3 g/d) and then remained unchanged on d 4 and 7. Therefore, Mg excretion was only transiently affected by an abrupt increase in dietary K whereas the effect of increased dietary Mg is more persistent. Treatment induced changes in MFA suggest both dietary Mg and K affected rumen ecology.