The domain of unknown function 560 (DUF560), also known as the PDDEXK_6 family, is a ubiquitous plant protein that has been confirmed to play critical roles in Arabidopsis root development as well as ABA and abiotic responses. However, genome-wide identification and expression pattern analysis in rice (Oryza sativa) still need to be improved. Based on the phylogenetic relationship, 10 OsDUF506 genes were identified and classified into four subfamilies. Segmental duplication was essential to the expansion of OsDUF506s, which were subjected to purifying selective pressure. Except for OsDUF50609 and OsDUF50610, the OsDUF506s shared colinear gene pairs with five monocot species, showing that they were conserved in evolution. Furthermore, the conserved domains, gene structures, SNPs distribution, and targeting miRNAs were systematically investigated. Massive cis-regulatory elements were discovered in promoter regions, implying that OsDUF506s may be important in hormone regulation and abiotic stress response. Therefore, we analyzed plant hormone-induced transcriptome data and performed qRT-PCR on eight OsDUF506s under drought, cold, and phosphorus-deficient stresses. The results revealed that most OsDUF506s respond to ABA and JA treatment, as well as drought and cold conditions. In conclusion, our findings provided insights into the evolution and function of OsDUF506s, which could benefit crop breeding in the future.