We report a first study of the semileptonic decay D⁰→ K⁻π⁰μ⁺νμ by analyzing an e⁺e⁻ annihilation data sample of 7.9~fb⁻¹ collected at the center-of-mass energy of 3.773 GeV with the BESIII detector. The absolute branching fraction of D⁰→ K⁻π⁰μ⁺νμ is measured for the first time to be (0.729 ± 0.014ₛₜₐₜ ± 0.011syst)%. Based on an amplitude analysis, the S- wave contribution is determined to be (5.76 ± 0.35ₛₜₐₜ ± 0.29syst)% of the total decay rate in addition to the dominated K*(892)⁻ component. The branching fraction of D⁰→ K*(892)⁻μ⁺ν_μ is given to be (2.062 ± 0.039ₛₜₐₜ ± 0.032syst)%, which improves the precision of the world average by a factor of 5. Combining with the world average of B(D⁰→ K*(892)⁻e⁺νₑ), the ratio of the branching fractions obtained is B(D⁰→ K*(892)⁻μ⁺ν_μ) B(D⁰→ K*(892)⁻e⁺νₑ) = 0.96±0.08, in agreement with lepton flavor universality. Furthermore, assuming single-pole dominance parameterization, the most precise hadronic form factor ratios for D⁰→ K*(892)⁻ μ⁺ν_μ are extracted to be rV=V(0)/A₁(0)=1.37 ± 0.09ₛₜₐₜ ± 0.03syst and r₂=A₂(0)/A₁(0)=0.76 ± 0.06ₛₜₐₜ ± 0.02syst.
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