This study investigated the effect of cation optimization by mixed cultures of Neurospora crassa and Candida utilis on the true protein (TP) content. Firstly, to enhance the nutritional contents of rice straw (RS), two fermentation parameters (effect of inoculation ratio and inoculation time) were optimized. It was found that when C. utilis was inoculated 60 h later than N. crassa with the inoculation ratio of 1:5 (N. crassa to C. utilis), the maximum TP yield was obtained. In order to further optimize TP content, Plackett-Burman design (PBD) and Box-Behnken design (BBD) of response surface methodology (RSM) were adopted. The results of PBD indicated that Mn2+, Zn2+, and Cu2+ were the significant variables. The optimum values for the three cations determined by the BBD were as follows: Mn2+ 0.06 g/L, Zn2+ 0.15 g/L, and Cu2+ 0.2 g/L. After the optimization of RSM, a model was proposed to predict the optimum value 10.36% confirmed by the experimental result 9.84%. The TP content increased from 3.98 to 9.84%, with 147.24% of its increase rate. This study proposed an ecofriendly and economical way to convert RS into protein-enriched livestock feed.