Aim Six1 is necessary for the genesis of several tissues, but in adults, it is expressed primarily in skeletal muscle where its function is unclear. Overexpression of Six1 with a cofactor in skeletal muscle causes slow‐to‐fast fibre‐type transition. We sought to characterize the effects of a physiologically relevant Six1 knockdown. Methods The tibialis anterior (TA) muscles of C57BL/6 mice were electroporated with Six1 knockdown vector (siRNA) or empty vector. Muscles were collected at 2 or 14 days after transfection for Six1 and myosin heavy chain (MHC) expression analysis. C2C12 mouse myoblasts were grown in standard conditions. Cells were cotransfected with MHC promoter vectors and Six1 expression vectors. Cells were harvested after 4 days of differentiation. Results In vivo, the Six1 siRNA caused a decreased expression of Six1,1.8‐fold (±0.1, P < 0.05). With decreased Six1, MHC IIB expression decreased 2.7‐fold (±0.7, P = 0.04). Proportion of muscle fibres expressing MHC IIB decreased (45.3 ± 4.8% vs. 65.1 ± 7.3% in control group, P = 0.04), and total area expressing MHC IIB decreased with decreased Six1 (59.6 ± 4.3% vs. 75.2 ± 5.4% in control group, P < 0.05). Decreased Six1 increased MHC IIA expression 1.9‐fold (±0.3, P = 0.04). In vitro, Six1 overexpression increased promoter activation of MHC IIB 2.9‐fold (±0.3, P < 0.01). Six1 knockdown repressed MHC IIB promoter 2.9‐fold (±0.1, P < 0.05) and MHC IIX 3.7‐fold (±0.08, P < 0.01). Conclusion Six1 knockdown caused a fast‐to‐slow shift in MHC isoform, and this was confirmed by promoter activity of MHC genes. Six1 may ultimately control the contractile and metabolic properties that define muscle fibre phenotype.