Humic acid and brassinosteroid applications may be an alternative to decrease the pineapple plantlet acclimatization in in vitro cultivation, since promising results have been observed when these substances were independently applied in other propagation methods. In this sense, the aim of the present study was to evaluate the effects of humic acids and brassinosteroid application on 'BRS Vitória' pineapple plantlets grown from in vitro cultivation during acclimatization. A randomized block design was used in a 5x2x4 factorial scheme, at five brassinosteroid doses (0, 0.25, 0.50, 0.75, 1.0 mg L-1) in the presence and absence of humic acids during four sampling periods (60, 90, 120 and 150 days after transplanting), with five replicates for each treatment. BIOBRÁS-16 was used as the brassinosteroid source, and the organic soil conditioner Agrolmin HF ® was used as the humic acid source. Plantlets were collected for evaluation every 30 days from 60 days after transplanting. The number of plantlet leaves, length and root mass were higher in the humic acid treatment without brassinosteroid application. Leaf, nitrogen, phosphorus, potassium, calcium and magnesium contents were of 13.04, 1.77, 40.2, 8.79 and 3.17 mg kg-1 , respectively. Nitrogen and potassium contents in the plantlets decreased, while phosphorus contents increased as a function of acclimatization time, regardless of treatment.