Conversion of energy-storing white adipose tissue into energy-burning beige adipose tissue, called browning, has emerged as a promising approach in the field of metabolic research and obesity treatment. Adeno-associated viruses (AAV) are widely used as viral vectors for gene delivery in eucaryotic cells. This study focused on the efficacy of transduction of AAV 2/5, 2/6, 2/8 expressing GFP in 3T3-L1 murine preadipocyte cells by live imaging microscopy using IncuCyte S3. Three transduction modes were assessed: AAVs transduction in 3T3-L1 preadipocyte cells with or without further differentiation into mature adipocyte-like cells and injection of AAVs in differentiated adipocyte-like cells. The differentiation of 3T3-L1 was induced by adipogenic IBMX-DEX-INS cocktail. AAV2/6 demonstrated the highest transduction efficiency in 3T3-L1 preadipocytes, as it was 1.5–2-fold more effective than AAV2/5 and AAV2/8 in the range of viral concentration from 2×10⁴ to 8×10⁴ VG/cell. AAV2/5 and AAV2/8 showed the transduction efficiency similar to each other. The expression of GFP under CMV promoter remained stable up to 20 days. The induction of 3T3-L1 differentiation in three days after AAVs transduction didn’t alter the GFP expression level and AAV2/6 showed the highest transduction efficiency. AAV2/6 demonstrated ability to transduce mature adipocytes as well. Thus, AAV2/6 compared to AAV2/5 and AAV2/8 demonstrated the higher transduction efficacy in 3T3-L1 preadipocytes and mature adipocytes, which proved its usability along with AAV8 and AAV9 for gene delivery to adipocytes.