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Ammonium (NH₄⁺) and nitrate (NO₃^&#8722;) are the two forms of inorganic nitrogen essential for physiological and biochemical processes in higher plants, but little is known about how the NH₄⁺:NO₃^&#8722; ratio may affect nitrogen metabolism. This study determined the effect of NH₄⁺:NO₃^&#8722; ratios on plant growth, accumulation, and distribution of nutrient elements, fruit quality, enzyme activity, and relative expression of genes involved in nitrogen (N) metabolism in pepper (<i>Capsicum</i> <i>annuum</i> L.). In a pod experiment, the NH₄⁺:NO₃^&#8722; ratios of 0:100, 12.5:87.5, 25:75, 37.5:62.5, and 50:50 were arranged in a complete randomized design with three replicates. The application of NH₄⁺:NO₃^&#8722; at 25:75 resulted in highest dry matter and N, phosphorus (P), and potassium (K) accumulation. Pepper treated with 25:75 ratio increased root length, surface areas, and root volume and tips. The contents of vitamin C, soluble sugar, soluble protein, total phenols, flavonoids, and capsaicinoids in the fruits were significantly higher with the NH₄⁺:NO₃^&#8722; ratio of 25:75 compared with 0:100 treatment, while lowering nitrate content was found in NH₄⁺:NO₃^&#8722; ratios of 25:75, 37.5:62.5, and 50:50 treatments. Activity of glutamine synthetase (GS), glutamate synthases (GOGAT) enzyme and the levels of relative expression of genes coding these enzymes were superior when the NH₄⁺:NO₃^&#8722; ratio of 25:75 were applied. Therefore, an appropriate ratio of NH₄⁺:NO₃^&#8722; (25:75) in nitrogen application can stimulate root development, promote enzyme activities, and enhance the productivity and fruit quality in pepper.