Heukharang (Lactuca sativa L.), the artificially developed lettuce cultivar for enhancement of lactucin content, exhibits antioxidant activities and sleep-promoting effects. However, potential of Heukharang as a raw material for the skin-whitening agent has not been investigated yet. This study evaluated the effects of Heukharang extract (HHE) on α-melanocyte-stimulating hormone (α-MSH)-induced melanogenesis in vitro and in vivo models. Our findings revealed that HHE (25–100 μg/mL) effectively inhibited α-MSH-induced melanin synthesis in both 2D and 3D cell culture environments without compromising the viability of the B16F10 murine melanoma cell line. Furthermore, HHE suppressed intracellular tyrosinase activity, along with both gene and protein expression of tyrosinase (TYR) and tyrosinase-related protein (TRP)-1 and TRP-2. HHE downregulated the mRNA level of microphthalmia-associated transcription factor (MITF) and the phosphorylation of cAMP response element-binding (CREB) protein. The antimelanogenic activity of HHE (50 and 100 μg/mL) was also confirmed using a zebrafish embryo model, highlighting its efficacy in inhibiting α-MSH-induced melanogenesis through the downregulating CREB/MITF/TYR signaling pathways. Moreover, our study demonstrated that a corresponding amount of lactucin (5–20 μM) to the HHE inhibited and regulated melanogenesis. These results collectively suggest that HHE, enriched with lactucin, holds promise as a potential skin-whitening agent for nutricosmetic industries.