Heavy metal contamination is one of the current serious environmental and agricultural soil issues, and it is mainly due to anthropogenic activities. Vanadium (V) is found in low concentrations in a wide range of plants and is widely distributed in soils. The current study aimed to determine how pepper seedlings responded to various V concentrations, as well as the detrimental effects of V on growth, root morphological traits, photosynthetic performance, reactive oxygen species (ROS), osmolytes production, antioxidant enzyme activities, and V uptake. Pepper seedlings (5 weeks old) were grown in hydroponic culture with six V levels (0, 10, 20, 30, 40, and 50 mg L−1 NH4VO3). After two weeks of V treatment, low level of V (10, 20 mg L−1) enhanced the growth status, conversely higher V (30, 40, and 50 mg L−1) level reduced the growth. The leaf gas exchange elements, pigments molecules, and root growth characteristics are also affected by higher V concentrations. Moreover, V uptake was higher in roots than in the shoot of pepper seedlings. Similarly, osmolytes content, ROS production, and antioxidant enzymes activities were significantly improved under V stress. Concluding, lower V (10, 20 mg L−1) concentration positively affected pepper growth, and higher V (30, 40, and 50 mg L−1) concentration had a detrimental effect on pepper physiological and biochemical mechanisms.