With the growth of cultural diversity in online higher education, this qualitative research aimed to investigate instructors' perceptions, practices, and challenges of increasing cultural presence in online course design and delivery. Seventeen instructors with different disciplinary backgrounds, online teaching experiences, titles, and ranks participated in semi-structured interviews. The research findings showed that instructors experienced multifaceted opportunities and challenges while developing culturally inclusive online learning environments. Four themes emerged regarding their perceptions and practices, including (1) cultural sensitivity and intersectionality in learner analysis and empathy, (2) developing culturally responsive online learning experiences, (3) humanizing online instruction for relationship and community building, and (4) decolonizing power dynamics to advance divergent voices and thinking. Instructors also encountered some difficulties and barriers: (1) the complexity of online instruction, (2) the lack of organic socioemotional connection, (3) the heavy workloads, and (4) the limitations of resources and support tailored to online learners.