Ca2+ is a ubiquitous second messenger which plays essential roles in many cellular processes, including cell differentiation. Store-operated Ca2+ entry (SOCE), a major calcium regulation mechanism in non-excitable cells, tightly regulates dynamic Ca2+ signals. Stromal interaction molecule (STIM) senses ER Ca2+ depletion and induces Ca2+ influx by activating Orai Ca2+ channel located in the plasma membrane. Recently, we reported that STIM2 splicing variant plays a role in myogenesis. However, whether this STIM2 splicing variant is involved in chondrogenic cell differentiation remains elusive. Here, we show that STIM2 splicing variant is essential for chondrogenesis. Reverse transcription polymerase. chain reaction revealed increased expression of SOCE components in differentiating ATDC5 chondrogenic cells. To confirm the role of STIM2 splicing variant in chondrogenesis, we generated STIM2 splicing variant knock-out ATDC5 cell by CRISPR-Cas9 system. The deletion of STIM2 splicing variant showed the altered calcium level and delayed physiological processes such as proliferation, migration, and differentiation. These results provide the evidence that STIM2 splicing variant might play an essential role in chondrogenic ATDC5 cell differentiation. This study improves the understanding of cellular differentiation research, particularly mediated by SOCE.