The β-1,4 galactosylation catalyzed by β-1,4 galactosyltransferases (β4Gal-Ts) is not only closely associated with diverse physiological and pathological processes in humans but also widely applied in the N-glycan modification of protein glycoengineering. The loop-closing process of β4Gal-Ts is an essential intermediate step intervening in the binding events of donor substrate (UDP-Gal/Mn2+) and acceptor substrate during its catalytic cycle, with a significant impact on the galactosylation activities. However, the molecular mechanisms in regulating loop-closing dynamics are not entirely clear. Here, we construct Markov state models (MSMs) based on approximately 20 μs of all-atom molecular dynamics simulations to explore the loop-closing dynamics for β-1,4 galactosyltransferase 1 (β4Gal-T1). Our MSM reveals five key metastable states of β4Gal-T1 upon substrate binding, indicating that the entire conformational transition occurs on a time scale of ∼10 μs. Moreover, a regulatory mechanism involving six conserved residues (R187, H190, F222, W310, I341, and D346) among β4Gal-Ts is validated to account for the loop-closing dynamics of the C-loop and W-loop by site-directed mutagenesis and enzymatic activity assays, exhibiting high consistency with our computational predictions. Overall, our research proposes detailed atomic-level insight into the loop-closing dynamics of the C-loop and W-loop on β4Gal-T1, contributing to a deeper understanding of catalytic mechanisms of β-1,4 galactosylation.