Optimal plant growth in many species is achieved when the two major forms of N are supplied at a particular ratio. In this pot experiment, the effects of five different ammonium:nitrate ratios (ANRs) (0:100, 12.5:87.5, 25:75, 37.5:62.5, and 50:50) on photosynthesis efficiency in chilli pepper (Capsicum annuum L.) plants were evaluated. The results showed that an ANR of 25:75 increased the contents of chl a, leaf area and dry matter, whereas chl b content was not affected by the ANRs. Regarding chlorophyll fluorescence, an ANR of 25:75 also enhanced the actual photochemical efficiency, photochemical quenching and maximum photosynthetic rate. However, the 0:100 and 50:50 ANRs resulted in higher values for nonphotochemical quenching. An inhibition of maximal photochemical efficiency was found when 50% NH4+ was supplied at the later stage of plant growth. The addition of 25% or 37.5% NH4+ was beneficial for gas exchange parameters and the 25% NH4+ optimised the thylakoid of chloroplasts. Compared with nitrate alone, 12.5-50% NH4+ upregulated glutamate dehydrogenase (GDH), the large subunit and the small subunit of Rubisco. It can be concluded that the 25:75 ANR accelerated N assimilation through active GDH, which provides a material basis for chloroplast and Rubisco formation, resulting in the increased photosynthetic rate and enhanced growth in chilli pepper.