The presence of antimony (Sb) in high concentrations in the environment is recognized as an emerging problem worldwide. The toxicity of Sb in plant tissues is known; however, new methods of plant tolerance improvement must be addressed. Here, poplar callus (Populus alba L. var. pyramidallis) exposed to Sb(III) in 0.2 mM concentration and/or to silicon (Si) in 5 mM concentration was cultivated in vitro to determine the impact of Sb/Si interaction in the tissue. The Sb and Si uptake, growth, the activity of superoxide dismutase (SOD), catalase (CAT), guaiacol-peroxidase (G-POX), nutrient concentrations, and the concentrations of photosynthetic pigments were investigated. To elucidate the action of Si during the Sb-induced stress, the impact of short and long cultivations was determined. Silicon decreased the accumulation of Sb in the calli, regardless of the length of the cultivation (by approx. 34%). Antimony lowered the callus biomass (by approx. 37%) and decreased the concentrations of photosynthetic pigments (up to 78.5%) and nutrients in the tissue (up to 21.7%). Silicon supported the plant tolerance to Sb via the modification of antioxidant enzyme activity, which resulted in higher biomass production (increased by approx. 35%) and a higher uptake of nutrients from the media (increased by approx. 10%). Silicon aided the development of Sb-tolerance over the longer cultivation period. These results are key in understanding the action of Si-developed tolerance against metalloids.