The polymerization and severing of microtubules are fundamental to the growth and branching of neurites in hippocampal neurons. The catalytic ATPase-containing A-subunit of katanin p60 (p60, KATNA1) promotes growth and development of hippocampal neurites by severing microtubules, while collapsing response mediator protein 3 (CRMP3) assembles microtubules to regulate neurite outgrowth. However, whether microtubule severing and assembling proteins would work together to regulate neurite outgrowth, especially for KATNA1 and CRMP3 remains to be elucidated. In this study, we revealed the interaction between KATNA1 and CRMP3 through GST-pulldown and co-immunoprecipitation assays and identified the binding domains between KATNA1 and CRMP3 as the MIT of KATNA1 (residues 1-77) and the D region of CRMP3 (residues 64-413). Furthermore, we demonstrated that CRMP3 enhances the microtubule-severing efficiency of KATNA1. In cultured hippocampal neurons, overexpression of KATNA1 and CRMP3 increased neurite length and branch number, and co-expression of both proteins further enhanced the promoting effect. Moreover, genetic knockout of KATNA1 or/and CRMP3 significantly inhibited neurite outgrowth. Overall, our data suggest that the CRMP3 interaction enhances the severing activity of KATNA1, thereby promoting hippocampal neurite outgrowth.