Abstract: The L-shape mitochondrial complex I (CI) consists of four modules: nicotinamide-adenine dinucleotide hydrogen-binding module (N) and ubiquinone-binding module (Q) in the matrix arm and proximal-pump module (P_P) and distal-pump module (P_D) in the membrane arm. As mitochondrial mutants are unavailable, the CI assembly pathway in plants is unclear. We investigated the CI assembly process using the maize RNA processing mutants deficient in individual CI components. Complexome profiling detected all major assembly intermediates of each module, confirming their independent assembly pathway. A block in the Q module assembly causes the accumulation of the membrane arm, whereas a block in the P_D module assembly results in the accumulation of CI*, a subcomplex assembled by P_P with the matrix arm. We further isolated and analyzed the mutants of two CI assembly factors, ZmGLDH and CRK1. The absence of either ZmGLDH or CRK1 eliminates the accumulation of CI* but allows a substantial amount of CI to be assembled. The membrane arm was also accumulated in zmgldh and crk1. Together, these results suggest two CI assembly pathways. The P_D-early former starts with the assembly of P_P with P_D, forming the membrane arm, then joins with the matrix arm to produce CI. The P_D-late pathway is initiated by assembling P_P with the matrix arm to yield CI*, which is then combined with P_D to form CI.