Insulin-loaded calcium pectinate nanoparticles were prepared as a potential colonic delivery system by ionotropic gelation with calcium ions. The effects of pectin molecular weight (M-v) and formulation pH on the characteristics of the nanoparticles were evaluated. Commercial pectins, LM101 and LM104, with respective degrees of esterification of 36% and 28%, were depolymerized by mechanical milling to give Mv ranging from 89 to 5.6 kDa. Milled pectins did not yield nanoparticles with significantly different mean diameter and insulin association efficiency (AE) compared to nanoparticles of umnilled pectins. LM104 nanoparticles had smaller variation in mean size than the LM101 nanoparticles. Formulation pH significantly influenced the AE and stability of the nanoparticles. Increasing the pH from 2 to 3 enhanced the AE by three-fold, from 32.76% to 93.31%, at an insulin loading concentration of 80 U/mL. This increase in AE was correlated to the charge density on the pectin molecules as a function of pH. Subsequent release of associated insulin from the nanoparticles was dependent on the extent of dilution of the nanoparticle dispersion and the pH of the dissolution medium. Cross-flow filtration could be used to separate the nanoparticles from unassociated ions and molecules, without compromising the characteristics of the nanoparticles.