The current research was carried out to investigate the effects of iron source in the culture media for Vaccinium corymbosum L. ʻBluerayʼ, ʻDukeʼ, and ʻPatriotʼ cultivars grown on five different types of medium (Woody Plant Medium supplemented with 1.0 mg·L−1 zeatin and 0, 25, 50, 75, and 100 mg·L−1 Sequestrene 138). After 10 weeks of culture, seven physiological parameters were measured, such as the number and length of axillary shoots, rooting and acclimatization percentage, as well as chlorophyll (a, b, a/b) and carotenoid content of the leaves. Adding Sequestrene 138 to the culture media led to a slight decrease of the proliferation rate but increased the length of the shoots. The chlorophyll and carotenoid content in all of the three cultivars was considerably increased as the iron concentration of the media increased. The shoots developed on the Sequestrene 138–free medium were chlorotic and short, whereas at different concentrations of iron in the culture medium the shoots were dark green and vigorous, providing a greater acclimatization success than those grown in iron-free medium.