Background: Shikonin, a natural compound derived from Lithospermum erythrorhizon, exhibits anticancer properties by inducing apoptosis in various tumor types, including clear cell renal cell carcinoma (ccRCC) cell lines CAKI-2 and A-498. This study investigates the mechanisms underlying shikonin-induced apoptosis, focusing on microRNAs miR-15b, miR-99b, and miR-181a in ccRCC. Materials and Methods: ccRCC cells were treated with 5 µM shikonin. Expression levels of miR-15b, miR-99b, and miR-181a were measured by TaqMan PCR. Apoptosis-related targets (AKT3, PDCD4, FOXO1, FOXO3, JNK1, and LAMTOR3) were identified in silico and validated by qRT-PCR and Western blot. Spearman’s correlation was used to evaluate miRNA–target relationships. Ingenuity Pathway Analysis explored relevant pathways. Results: Shikonin decreased miR-15b, miR-99b, and miR-181a levels in CAKI-2 cells, whereas these miRNAs were increased in A-498 cells, demonstrating cell-line-specific effects. qRT-PCR and Western blot confirmed changes in target expression, suggesting regulation by these miRNAs. In A-498 cells, miR-181a expression positively correlated with the studied target levels during 24–72 h of treatment, indicating that its potential regulatory role may be cell-type-dependent. MiR-15b and miR-99b showed linear correlations with targets in both cell lines, but expression patterns differed, suggesting direct regulation alongside potential involvement in additional pathways contributing to shikonin-induced apoptosis. Conclusions: Shikonin induces apoptosis in renal cancer cells by modulating the MAPK/ERK pathway and through cell-line-specific, cell-type-dependent regulation of miR-15b, miR-99b, and miR-181a. These findings highlight the importance of cell-type-dependent miRNA regulation and underscore the therapeutic potential of shikonin in ccRCC.