Singlet fission has the great potential to overcome the Shockley–Queisser thermodynamic limit and thus promotes solar power conversion efficiency. However, the current limited understandings of detailed singlet fission mechanisms hinder a further improved design of versatile singlet fission materials. In the present study, we combined ultrafast transient infrared spectroscopy with ab initio calculations to elucidate the roles played by the vibrational normal modes in the process of singlet fission for hexacene. Our transient infrared experiments revealed three groups of vibrational modes that are prominent in vibronic coupling upon photoexcitation. Through our computational study, those normal modes with notable Franck-Condon shifts have been classified as ring-twisting modes near 1300.0 cm⁻¹, ring-stretching modes near 1600.0 cm⁻¹, and ring-scissoring modes near 1700.0 cm⁻¹. Experimentally, a ring-stretching mode near 1620.0 cm⁻¹ exhibits a significant blue-shift of 4.0 cm⁻¹ during singlet fission, which reaction rate turns out to be 0.59 ± 0.07 ps. More interestingly, the blue-shifted mode was also identified by our functional mode singlet fission theory as the primary driving mode for singlet fission, suggesting the importance of vibronic coupling when a correlated triplet pair of hexacene is directly converted from its first excited state singlet exciton. Our findings indicate that the ultrafast transient infrared spectroscopy, in conjunction with the nonadiabatic transition theory, is a powerful tool to probe the vibronic fingerprint of singlet fission.