We give a comprehensive study from flavor observables of π, K, D (s) , and B (s) mesons for limiting the Two Higgs Doublet Models (2HDMs) with natural flavor conservation, namely, Z 2 symmetric (type I, II, X, Y) and aligned types of models. With use of updated theoretical predictions and experimental analyses ofd mixing, and B 0 s -B 0 s mixing, we obtain constraints on the parameters in the 2HDMs. To calculate the constraints, we pay attention to a determination of CKM matrix elements and re-fit them to experimental data so that new contributions from additional Higgs bosons do not affect the determination. As a result, we find that the charged Higgs boson mass less than around 490 GeV is ruled out fromB → X s γ in the type II and Y models, whereas large tan β is excluded from B 0 s → µ + µ − in the type II. We also see that severe constraints on the mass and couplings are put fromB → X s γ, B 0 s → µ + µ − , and B 0 s -B 0 s in the aligned model. In addition, we discuss excesses of observables in the muon anomalous magnetic moment and the semi-tauonic B meson decays in the context of the 2HDM, and find that the aligned model can explain part of the excesses, compatible with the other constraints.