This study compared the in vitro and in vivo effects of different aliphatic acid grafted N-trimethyl chitosan (TMC) surface-modified nanostructured lipid carriers (NLC) by oral delivery. Medium-chain fatty acids, decylic acids (DA), and long-chain fatty acids, palmitic acids (PA) were selected as contrasting objects. TMC, DA grafted TMC (DA-TMC), and PA grafted TMC (PA-TMC) were successively synthesized. Kaempferol loaded NLC (KNLC), KNLC coated with DA-TMC (DA-TMC-KNLC) and PA-TMC (PA-TMC-KNLC) were fabricated, respectively. KNLC were subspherical in shape at nano-size limits. The particle size increased from 93.6 to 125.5 nm and the zeta potential changed from negative to positive due to surface-modification. The KNLC surface-modified with different aliphatic acid grafted TMC displayed a diverse release profiles at the simulative physiological environment, which contrasted that of KNLC. Pharmacokinetic studies demonstrated that the nanoparticles all could improve the AUC values and prolong blood retention times compared to that of kaempferol suspensions. Cell uptake and in situ intestinal perfusion experiments revealed that DA-TMC-KNLC and PA-TMC-KNLC could remarkably enhance cellular uptake of kaempferol into Caco-2 cells and drug absorption in each intestinal segment in comparison with KNLC, repectively. Wherein, DA-TMC-KNLC exhibits the greatest uptake and absorption efficiency as compared to kaempferol suspensions, KNLC and PA-TMC-KNLC. Collectively, DA-TMC surface-modified NLC might serve as a potential drug carrier for oral delivery of water-insoluble flavonoid ingredients.