© 2018, © 2018 Taylor & Francis. The water vapor permeability (WVP) and hardness of biodegradable nanocomposite films based on glutelin from Jatropha curcas L. were determined and the results were analyzed by response surface methodology (RSM). The effects of independent variables constituted by the plasticizer (glycerol), nanofiller (cloisite), and pH on the physical parameters were evaluated. The regression models obtained for each physical property adequately described the measurements used in the experimental design, with regression coefficients of 0.9513 and 0.9421 for WVP and hardness. The regression coefficients show that pH has the most significant effect on the WVP, whereas the cloisite content has the largest influence on the hardness of the nanocomposite film. Fourier transform infrared spectroscopy provided confirmation of the results obtained by RSM. Corroboration that the gluteline structure is in a similar form as the WVP and primarily influenced by pH was obtained using conventional and second derivative infrared spectra. However, the hardness of the film primarily depends on the cloisite content. This methodology may be useful for the design and elaboration of nanocomposite films based on J. curcas L. with specific barrier and mechanical properties.