Previously, we showed luteal stage-specific regulation of angiogenesis-modulating factors by prostaglandin F2 alpha (PGF2alpha). Fibroblast growth factor 2 (FGF2) and thrombospondins (THBSs) exhibited the most divergent profile of induction by PGF2alpha. We therefore examined the transcriptional regulation and roles of THBSs in luteal cells and studied their interaction with FGF2. THBSs and their receptors exhibited cell-specific expression: THBS1 was the predominant form in luteal endothelial cells (LEC), whereas luteinized granulosa cells (LGC) expressed mostly THBS2. CD36 was confined to LGC, but CD47 did not exhibit preferential expression between LEC and LGC. THBS1 and THBS2 were both stimulated in vitro by PGF2a and its analog in LGC. In contrast, luteinizing signals (LH and insulin) decreased the expression of THBS1, THBS2, and CD36. Importantly, LH increased FGF2 expression, suggesting that THBSs and FGF2 are conversely regulated. We found that FGF2 inhibited THBS1 and vice versa, and that THBS1 treatment decreased FGF2 expression, suggesting reciprocal inhibition. In agreement, ablation of THBS1 by specific small interference RNAs elevated FGF2 levels. THBS1 reduced LEC numbers and promoted apoptosis by activation of caspase-3. In contrast, FGF2 reduced basal and THBS1-induced caspase-3 levels. Consistent with these findings, small interference RNA silencing of THBS1 in luteal cells reduced the levels of active caspase-3 and improved the survival of cells when challenged with staurosporine. Taken together, these studies suggest that THBSs are suppressed during luteinization but are induced by PGF2alpha in luteolysis. THBS1 has antiangiogenic, proapoptotic properties; these, together with its ability to inhibit FGF2 expression and activity, can promote luteolysis.