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Using 20.3 fb-1 of e+e- collision data collected at the center-of-mass energy 3.773 GeV with the BESIII detector, we report the first observation of the semileptonic decay D+→η′μ+νμ with significance of 8.6σ including systematic uncertainties, and an improved measurement of D+→η′e+νe. The branching fractions of D+→η′μ+νμ and D+→η′e+νe are determined to be (1.92±0.28stat±0.08syst)×10-4 and (1.79±0.19stat±0.07syst)×10-4, respectively. The ratio of the two branching fractions is determined to be Rμ/e=1.07±0.19stat±0.03syst, which agrees with the theoretical expectation of lepton flavor universality within the standard model. From an analysis of the D+→η′ℓ+νℓ decay dynamics, the product of the hadronic form factor f+η′(0) and the Cabibbo-Kobayashi-Maskawa matrix element |Vcd| is measured for the first time, giving f+η′(0)|Vcd|=(5.92±0.56stat±0.13syst)×10-2. The η-η′ mixing angle in the quark flavor basis is determined to be φP=(39.8±0.8stat±0.3syst)°.