Left-right alternation depends on activity in commissural interneurons (CINs) that have axons crossing in the midline. In this study, we investigate the CIN connectivity to local motor neurons using a newly developed preparation of the in vitro neonatal mouse spinal cord that allows us to identify all classes of CINs. Nineteen of 29 short-range CINs with axonal projections <1.5 segments (sCINs) directly excited, directly inhibited, or indirectly inhibited contralateral motor neurons in the quiescent spinal cord. Excitation was glutamatergic and inhibition was mixed glycinergic and/or GABAergic. Long-range CINs were also found to have input to local, contralateral motor neurons. Thirteen of 29 descending CINs had similar synaptic connectivity to contralateral motor neurons as the sCINs, including direct excitation and direct and indirect inhibition. Some (9 of 23) rostrally projecting ascending CINs, and a few (2 of 10) CINs with bifurcating axons that both ascend and descend, indirectly inhibited local, contralateral motor neurons. Rhythmic firing during locomotor-like activity was observed in a number of CINs with segmental synaptic effects on contralateral motor neurons. This study outlines the basic connectivity pattern of CINs in the mouse spinal cord on a segmental level. Our study suggests that, based on observed synaptic connectivity, both short- and long-range CINs are likely involved in segmental left-right coordination and that the CIN system is organized into a dual-inhibitory and single-excitatory system. These systems are organized in a way that they could provide appropriate coordination during locomotion.