This paper reports both the optimization of aconitine entrapment and its release from biodegradable poly(d,l-lactide-coglycolide) (PLGA) nanoparticles prepared using the O/W single-emulsion/solvent-evaporation technique. The influence of several parameters, such as the initial aconitine mass, aqueous-phase pH, volume ratio of aqueous/organic phase (W/O), PLGA concentration in the organic phase, etc., on aconitine encapsulation were investigated. The optimized nanoparticles had an entrapment efficiency of 88.40+/-3.02% with drug loading capacity of 9.42+/-2.93%. Crystallization growth, which played a crucial role in hindering the incorporation of aconitine into the polymer carrier, was proposed. Differential scanning calorimetry and X-ray powder diffraction demonstrated that aconitine existed in an amorphous state or as a solid solution in the polymeric matrix. The in vitro release profiles exhibited a sustained release of aconitine from nanoparticles and a pH-dependent character in phosphate-buffered saline with different pH values. Moreover, aconitine-loaded PLGA nanoparticles could lead to improvement in the stability of aconitine. This work demonstrated the feasibility of encapsulating aconitine into PLGA nanoparticles using the O/W single-emulsion/solvent-evaporation technique.