Sphingomyelin synthase 1 (SMS1) and 2 (SMS2) are two enzymes required for sphingomyelin de novo synthesis, and their roles in tumor transformation and development have been recently recognized. In this work, we systematically evaluated the expression patterns of SMS1 and 2 in ovarian cancer patient samples and cell lines. Furthermore, we analyzed the functions of SMS2 and its underlying mechanisms. We observed a specific increase in SMS2 expression in ovarian cancer tissues compared to the adjacent normal ovary tissues in majority of patients' samples. This is regardless of their clinico-pathological characteristics. SMS1 expression was similar between ovarian cancer and its normal counterpart in 30 patients tested. The upregulation of SMS2 but not SMS1 was also reproducible in a panel of ovarian cancer cell lines. Functional analysis indicated that SMS2 plays a predominant role in promoting migration rather than proliferation in ovarian cancer. SMS2 depletion suppressed migration, growth and survival, and furthermore this was dependent on SMS2 baseline level in ovarian cancer cells. SMS2 inhibition significantly augmented cisplatin's efficacy. We further found that migration inhibition induced by SMS2 depletion was largely due to the suppression of RhoA/ROCK/LIMK/cofilin and RhoA/ROCK/FAK/paxillin pathways. In addition, lipid metabolism disruption, oxidative stress and damage, and impaired mitochondrial function contributed to the inhibitory effects of SMS2 depletion in ovarian cancer growth and survival. Our work demonstrates that SMS2 but not SMS1 is upregulated in ovarian cancer and involved in migration, growth and survival via different mechanisms. Our findings highlight the therapeutic value of SMS2 inhibition in the treatment of ovarian cancer.