Although nitrogen (N), phosphorus (P), and potassium (K) co-application improves crop growth, yield, and N use efficiency (NUE) of rice, few studies have investigated the mechanisms underlying these interactions. To investigate root morphological and physiological characteristics and determine yield and nitrogen use parameters, rhizo-box experiments were performed on rice using six treatments (no fertilizer, PK, N, NK, NP, and NPK) and plants were harvested at maturity. The aboveground biomass at the elongating stage and grain yield at maturity for NPK treatment were higher than the sum of PK and N treatments. N, P, and K interactions enhanced grain yield due to an increase in agronomic N use efficiency (NAE). The co-application of N, P, and K improved N uptake and N recovery efficiency, exceeding the decreases in physiological and internal NUE and thereby improving NAE. Increases in root length and biomass, N uptake per unit root length/root biomass, root oxidation activity, total roots absorption area, and roots active absorption area at the elongating stage improved N uptake via N, P, and K interactions. The higher total N uptake from N, P, and K interactions was due to improved root characteristics, which enhanced the rice yield and NUE.