Abstract: Pineapple Ananas comosus (Linn.) Merr. is China’s third largest tropical fruit, with the largest planting area in Xuwen County, Guangdong Province. As one of the most important macronutrients, nitrogen is closely related to pineapple yield. However, the uptake preferences for different nitrogen forms in field-grown pineapple plants remain unclear. In this study, the morphological, physiological, and growth traits of plants with different ages were measured in two field-grown pineapple cultivars (‘Tainong 17’ and ‘Bali’) with different growth periods in April and September, respectively, in Xuwen County. In addition, nitrogen acquisition strategies for three different forms of nitrogen (ammonium nitrogen, nitrate nitrogen, and glycine) in the pineapple roots were determined using the stable isotope ¹⁵N tracer technique. The results indicated that the growth period of the ‘Tainong 17’ pineapple (16 months) was shorter than that of ‘Bali’ (20 months). During the fruit harvest period in April, compared with the ‘Bali’ pineapple (796 g fresh fruit weight per plant), ‘Tainong 17’ pineapple plants had lower yield (532 g fresh fruit weight per plant), root biomass, and P content; but had similar plant height, plant biomass per plant, leaf N and K contents, and specific leaf area. As an indicator of long-term water-use efficiency, the δ¹³C value ranging from −15.16‰ to −13.28‰, was higher in the leaves of ‘Tainong 17’ pineapple than that in ‘Bali’. Neither cultivar nor age greatly affected the leaf δ¹³C values. In April and September, there were significant differences in the different forms of nitrogen uptake between the two pineapple cultivars. The nitrogen uptake capacity of ‘Tainong 17’ pineapple was higher than that of ‘Bali’. The high acquirement capacity of nitrogen and water use efficiency of ‘Tainong 17’ pineapple is attributed to promoting photosynthesis and thus maintaining plant growth in a relatively short life cycle. Both pineapple cultivars preferred to acquire ammonium nitrogen (36.8%–64.6%), followed by glycine (23.2%–47.1%), and the uptake rate of nitrate nitrogen was the lowest (9.1%–31.5%). The nitrogen uptake rate of pineapple plants in the vegetative growth stage (5–8-month-old) was higher than that of plants in the fruit-harvesting stage. However, with increasing plant age, the contribution rate of ammonium nitrogen increased, whereas that of glycine gradually decreased. Across different pineapple cultivars and plant ages, the rates of different forms of nitrogen uptake were not linearly correlated with the soil nitrogen content or measured plant traits. To the best of our knowledge, this is the first study to show that the roots of field-grown pineapple plants can directly absorb organic nitrogen from the soil. Cultivar and plant growth stages of pineapples are important factors that affect nitrogen acquisition strategies. However, the linear relationships between the absorption rates of different forms of nitrogen and soil nitrogen content or measured plant traits were very weak. These results contribute to nitrogen fertilizer management in pineapple plantations.