Proteins of the signal crayfish Pacifastacus leniusculus egg and spermatophore were identified using in-gel digestion, mass spectrometry, and Mascot search. Forty-one and one-hundred-fifty proteins were identified in egg and spermatophore, respectively. The proteins were classified into nine categories including cell defence, cell signaling, cytoskeleton, DNA related activity, metabolism and energy production, protease and protease inhibitor, respiration, transportation, and others and unknown. Twenty-two proteins were found in both egg and spermatophore. The respiration and cytoskeleton groups are the most diverse categories in the protein profiles of the egg and spermatophore, respectively. No protein was assigned to DNA related activity and cell defence categories in the protein profile of the crayfish egg. Differences between protein profiles of the crayfish egg and spermatophore show different functional priorities for each of gametes. Several proteins having possible roles in gametogenesis, capacitation, acrosome reaction, and fertilization were identified. This proteomic profile of signal crayfish gametes provides a basis for further investigation of functional roles of the identified proteins in aspects of reproduction such as capacitation and fertilization.